JP2004508385A - Mechanism of mitochondrial membrane permeabilization by HIV-1 VPR, a mimic of Vpr, and method for screening for active molecules capable of altering and / or preventing and / or mimicking the interaction between VPR and ANT - Google Patents

Abstract

The invention is directed to the induction of mitochondrial membrane permeabilization via the physical and functional interaction of the HIV-1 Vpr protein with the mitochondrial inner membrane protein ANT (adenine nucleotide translocator, also called adenine nucleotide translocase or ADP/ATP carrier). Reagents and methods for inducing and/or inhibiting the binding of Vpr to ANT, mitochondrial membrane permeabilization, and apoptosis are provided.

Description

【図面の簡単な説明】
【図１Ａ】
図１は、ＶｐｒとＡＮＴとの物理的および機能的な相互作用を表す。
Ａ．Ｖｐｒ５２〜９６、Ｖｐｒ５２〜９６〔Ｒ７３Ａ，８０Ａ〕または無関係な対照（Ｃｏ）とのＡＮＴの相互作用のプラスマ表面共鳴センサーグラム。Ｖｐｒ５２〜９６との相互作用のセンサーグラムだけが、時間の関数としての結合の増大、および解離期（ｏｆｆ）の開始時における正のシグナルを示す。相互作用の計算されたＫ_Ｄ（Ｋ_Ｄ＝ｋ_ｏｎ／ｋ_ｏｆｆ）は９．７±６．４ｎＭ（Ｘ±ＳＤ、ｎ＝５）である。
【図１Ｂ】
図１は、ＶｐｒとＡＮＴとの物理的および機能的な相互作用を表す。
Ｂ．ブランクを差し引くことによって補正されたセンサーグラムにおけるＡＮＴの種々の濃度で測定されたラングミュア等温式（センサーグラムはＶｐｒ５２〜９６〔Ｒ７３Ａ，８０Ａ〕で得られた）。
【図１Ｃ】
図１は、ＶｐｒとＡＮＴとの物理的および機能的な相互作用を表す。
Ｃ．ＡＮＴリガンドおよびＡＮＴ由来ペプチドによるＶｐｒ５２〜９６−ＡＮＴ相互作用の調節。測定は、ボンクレキン酸（ＢＡ、２５０μＭ）、アトラクチロシド（Ａｔｒ、５０μＭ）、ＡＮＴ１０４〜１１６ペプチドまたは３つの対照ペプチド（すべて５μＭ）の非存在下（φ）または存在下においてＡのように行われた。ＡＮＴ−２由来ペプチドのＡＮＴ１０４〜１１６〔ＤＫＲＴＱＦＷＲＹＦＡＧＮ〕および対照ペプチド（Ｃｏ．Ｉ：スクランブル型ＡＮＴ１０４〜１１６〔ＦＱＮＹＷＧＨＫＲＦＲＤＡ〕；Ｃｏ．ＩＩ：変異型ＡＮＴ１０４〜１１６〔ＤＧＨＫＱＦＷＧＹＦＡＧＮ〕；Ｃｏ．ＩＩＩ：ＡＮＴ関連のヒトリン酸輸送体タンパク質に由来するトポロジー的に等価なペプチド（ａａ１４９〜１６１）〔ＳＮＭＬＧＥＥＮＴＹＬＷＲ〕。活性化または阻害は、（１−ｋ_０ａ／ｋ_０）ｘ１００として計算された（式中、ｋ_０ａおよびｋ_０はそれぞれ、薬剤の存在下または非存在下における初速度である）。
【図１Ｄ】
図１は、ＶｐｒとＡＮＴとの物理的および機能的な相互作用を表す。
Ｄ．ビオチン化Ｖｐｒ５２〜９６に対するＡＮＴ１０４〜１１６の結合に関するラングミュア等温式（Ａで測定されたとき）。相互作用の計算されたＫ_Ｄは３５μＭである。
【図１Ｅ】
図１は、ＶｐｒとＡＮＴとの物理的および機能的な相互作用を表す。
Ｅ．ＡＮＴおよびＡＮＴ−２由来ペプチドＡＮＴ１０４〜１１６のトポロジーを示す概略図である。
【図２Ａ】
図２は、ＶｐｒとＡＮＴ含有リポソームとの物理的相互作用（Ａ、Ｂ）および機能的相互作用（Ｃ）を表す。
Ａ．ＡＮＴ−リポソームおよびリポソーム単独に対するＦＩＴＣ標識Ｖｐｒ５２〜９６結合の用量応答曲線。
【図２Ｂ】
図２は、ＶｐｒとＡＮＴ含有リポソームとの物理的相互作用（Ａ、Ｂ）および機能的相互作用（Ｃ）を表す。
Ｂ．ＢＡ（５０μＭ）の存在下または非存在下でのリポソーム単独、ＡＮＴ−プロテオリポソームに対するＦＩＴＣ−Ｖｐｒ５２〜９６（２μＭ）の結合。
【図２Ｃ】
図２は、ＶｐｒとＡＮＴ含有リポソームとの物理的相互作用（Ａ、Ｂ）および機能的相互作用（Ｃ）を表す。
Ｃ．ＶｐｒによるＡＮＴプロテオリポソームの透過化（Ｘ±ＳＤ、ｎ＝３）。リポソームは、４−メチルウンベリフェリルホスファート（４−ＭＵＰ）が負荷され、そしてＢＡ（５０μＭ）、ＡＤＰ（８００μＭ）および／または示されたペプチド（Ｂの場合と同じ、０．５μＭ、Ｖｐｒ５２〜９５と５分間プレインキュベーションされる）の存在下または非存在下、Ａｔｒ（２００μＭ）または示されたＶｐｒ由来ペプチド（１μＭ）に８０分間さらされた。その後、アルカリホスファターゼが加えられ、リポソームから放出された４−ＭＵＰを蛍光色素の４−メチルウンベリフェロン（４−ＭＵ）に変換し、Ｖｐｒ由来ペプチドにより誘導された４−ＭＵＰ放出の割合を「材料および方法」に記載されるように計算した。
【図３Ａ】
図３は、平面状脂質二重層におけるＶｐｒ５２〜９６およびＡＮＴの電気生理学的性質を表す。Ｖｐｒ５２〜９６（８０ｎＭ、＋１５０ｍＶ）、Ｖｐｒ５２〜９６（０．４ｎＭ、＋１００ｍＶ）、ＡＮＴ（１ｎＭ、＋１１０ｍＶ）およびＶｐｒ５２〜９６＋ＡＮＴ（０．４：１ｎＭ、＋１１５ｍＶ）の電流変動、ならびにコンダクタンスレベルの関連するヒストグラム（右）が示される。
Ａ．単一チャンネルレベルでのＡＮＴとＶｐｒ５２〜９６との共同的作用。合成膜内に取り込まれた後における、Ｖｐｒ５２〜９６（８０ｎＭ、＋１５０ｍＶ）、Ｖｐｒ５２〜９６（０．４ｎＭ、＋１００ｍＶ）、ＡＮＴ（１ｎＭ、＋１１０ｍＶ）およびＶｐｒ５２〜９６＋ＡＮＴ（０．４：１ｎＭ、＋１１５ｍＶ）の電流変動。単一チャンネルでの記録が、「Ｔｉｐ−Ｄｉｐ」技術を使用して行われた。示される記録は、少なくとも３回の独立した測定に対する代表例である。
【図３Ｂ】
図３は、平面状脂質二重層におけるＶｐｒ５２〜９６およびＡＮＴの電気生理学的性質を表す。Ｖｐｒ５２〜９６（８０ｎＭ、＋１５０ｍＶ）、Ｖｐｒ５２〜９６（０．４ｎＭ、＋１００ｍＶ）、ＡＮＴ（１ｎＭ、＋１１０ｍＶ）およびＶｐｒ５２〜９６＋ＡＮＴ（０．４：１ｎＭ、＋１１５ｍＶ）の電流変動、ならびにコンダクタンスレベルの関連するヒストグラム（右）が示される。
Ｂ．Ａで得られたコンダクタンスの統計学的分析。結果は、種々の電圧における電流変動として表された。コンダクタンス（γ；ピコシーメンス（ｐＳ）単位で）が、電流を電圧で除することによって計算される。
【図４Ａ】
図４は、Ｖｐｒにさらされた精製ミトコンドリアの酸化的性質を示す。
Ａ．示された薬剤を添加した後における酸素消費曲線。軌跡ａ：対照ミトコンドリア（前処理なし）。軌跡ｂ：１μＭのＶｐｒ５２〜９６で１０分間にわたり前処理されたミトコンドリア。軌跡に沿った数字は、１分あたり１ｍｇのタンパク質について消費されたＯ_２のｎｍｏｌである。
【図４Ｂ】
図４は、Ｖｐｒにさらされた精製ミトコンドリアの酸化的性質を示す。
Ｂ．１μＭのＶｐｒ由来ペプチドを添加した１０分後のＣＣＣＰ存在下での酸素消費を、オリゴマイシンを用いて測定された酸素消費（Ａのように測定）で除することによって計算された呼吸制御（ＲＣ）値（３回の測定値の平均値）。
【図５Ａ】
図５は内側対外側のミトコンドリア膜透過化を示す。
Ａ．ＮＡＤＨおよびＶｐｒ５２〜９６（１μＭ）を添加した後で行われた呼吸測定。軌跡に沿った数字は、１分あたり１ｍｇのタンパク質について消費されたＯ_２のｎｍｏｌである。Ｖｐｒにより刺激されたＮＡＤＨ非依存性のＯ_２消費がロテノンに対して完全に感受性であったことに留意すること。
【図５Ｂ】
図５は内側対外側のミトコンドリア膜透過化を示す。
Ｂ．ＮＡＤＨに対するＶｐｒ５２〜９６誘導の内膜透過化の速度論、および低下したシトクロームｃに対する外膜透過化の速度論。酸素消費が、２ｍＭのＮＡＤＨの存在下（黒四角）でＡの場合（軌跡Ｃ〜Ｄ）のように測定され、そしてシトクロームｃ（１５μＭ）の酸化（白丸）が、記載（Ｒｕｓｔｉｎら、１９９４）のように分光蛍光測定法によって測定された。シトクロームｃ酸化の１００％値は２．５ｍＭのラウリルマルトシドの添加によって測定された。
【図５Ｃ】
図５は内側対外側のミトコンドリア膜透過化を示す。
Ｃ．Ｖｐｒ５２〜９６により誘導されるΔΨ_ｍ喪失およびシトクロームｃ放出の速度論。精製されたミトコンドリアを１μＭのＶｐｒ５２〜９６で処理し、ΔΨ_ｍ感受性蛍光色素ＪＣ−１を使用して、低いΔΨ_ｍを有するミトコンドリアの割合の細胞蛍光測定法による測定に供した。同時に、シトクロームｃがミトコンドリアの上清において免疫検出された。
【図６Ａ】
図６は、ミトコンドリアに対するＶｐｒ作用のＢｃｌ−２媒介の阻害を示す。
Ａ．無傷な細胞において誘導されるＶｐｒ５２〜９６誘導のΔΨ_ｍ消失。ＣＯＳ細胞は、組換えヒトＢｃｌ−２（１０μＭ）、Ｋｏｎｉｇｓポリアニオン（ＰＡ、２μＭ）またはＰＢＳのみが顕微注入され、次いで１μＭのＶｐｒ５２〜９６の非存在下（Ｃｏ．）または存在下で３時間インキュベーションされ、そしてΔΨ_ｍ感受性色素ＪＣ−１（２μＭ；ΔΨ_ｍが高いミトコンドリアの赤色蛍光、ΔΨ_ｍが低いミトコンドリアの緑色蛍光）で染色された。
【図６Ｂ】
図６は、ミトコンドリアに対するＶｐｒ作用のＢｃｌ−２媒介の阻害を示す。
Ｂ．ＮＡＤＨに対してＶｐｒ誘導される内側のＭＭＰに対するＢｃｌ−２の作用。ミトコンドリアは未処理（Ｃｏ．）のままにされるか、またはＢｃｌ−２（０．８μＭ）もしくはＢＡ（１０μＭ）で（１０分）前処理された。精製されたミトコンドリアの酸素消費が、示されるようにコハク酸塩＋ＣＣＣＰまたはＮＡＤＨを添加した後、図５のように測定された。
【図６Ｃ】
図６は、ミトコンドリアに対するＶｐｒ作用のＢｃｌ−２媒介の阻害を示す。
Ｃ．単離されたミトコンドリアに対するＶｐｒの超構造的作用。電子顕微鏡写真は、０．８μＭのＢｃｌ−２または２μＭのＰＡ１０とのプレインキュベーション（５分間）を行った後、３μＭのＶｐｒ５２〜９６と５分間または１５分間にわたってミトコンドリアをインキュベーションした後に得られた。
【図６Ｄ】
図６は、ミトコンドリアに対するＶｐｒ作用のＢｃｌ−２媒介の阻害を示す。
Ｄ．精製されたミトコンドリアにおけるＶｐｒ５２〜９６誘導によるΔΨ_ｍ消失に対するＢｃｌ−２およびＰＡ−１０の作用。単離されたミトコンドリア（２００μｇタンパク質／ｍｌ）を、示された阻害剤（５μＭのＣｓＡ、５０μＭのＢＡ、０．８μＭのＢｃｌ−２、２μＭのＰＡ１０；５分〜１０分）とプレインキュベーションし、洗浄（１０分、６８００ｇ、２０℃）して、ΔΨ_ｍ感受性色素ＪＣ−１（２００ｎＭ、１０分）とインキュベーションし、そしてＶｐｒ５２〜９８（３μＭ、５分）にさらして、蛍光（５７０ｎｍ〜５９５ｎｍ）および粒子サイズ（ＦＳＣ）のフローサイトメトリー測定に供した。数字は、約１０^４イベントの中でのＪＣ−１高ミトコンドリアおよびＪＣ−１低ミトコンドリアの割合を示す。
【図６Ｅ】
図６は、ミトコンドリアに対するＶｐｒ作用のＢｃｌ−２媒介の阻害を示す。
Ｅ．種々のＶｐｒ由来ペプチドとインキュベーションした後のＪＣ−１低ミトコンドリアの頻度の定量（Ｘ±ＳＤ、ｎ＝５）。精製されたミトコンドリアを、ＰＴ緩衝液において、Ｂｃｌ−２（０．８μＭ）、Ｂｃｌ−２Δα_{５ ／ ６}（０．８μＭ）またはＢＡ（１０μＭ）の存在下または非存在下で１０分間プレインキュベーションし、そしてΔΨ_ｍ感受性色素ＪＣ−１（２００ｎＭ、１０分）とインキュベーションし、その後、３μＭのＶｐｒ５２〜９６（ｗｔ、示されるようなビオチン化体または修飾型）と５分間、または５μＭ〜１０μＭのＶｐｒ１〜９６、Ｖｐｒ１〜５１、Ｖｐｒ７１〜９６、Ｖｐｒ７１〜８２（ｗｔまたは示されるような修飾型）と１０分間インキュベーションし、最後に、Ｄのようにフローサイトメトリー分析に供した。
【図７Ａ】
図７は、Ｖｐｒ５２〜９６がミトコンドリアに結合することに対するＢｃｌ−２およびＰＡ−１０の差のある作用を表す。
Ａ．Ｖｐｒ５２〜９６は、ΔΨ_ｍ喪失を誘導する前にミトコンドリアと結合する。ミトコンドリアは染色されないままにされた（Ｃｏ．における挿入図）か、またはΔΨ_ｍ非感受性ミトコンドリア色素ＭｉｔｏＴｒａｃｋｅｒ（登録商標）グリーン（７５ｍＭ）に、単独（ＭＴＧ）で、もしくは０．５μＭのＦＩＴＣ−Ｖｐｒ５２〜９５（緑色蛍光）とともに、ΔΨ_ｍ感受性ミトコンドリア色素ＭｉｔｏＴｒａｃｋｅｒ（登録商標）レッド（ＣＭＸＲｏｓ；赤色蛍光）と組み合わせてさらされ、その後、フローサイトメトリーによる二色分析が行われた。数字は、それぞれ四連におけるミトコンドリアの割合を示す。
【図７Ｂ】
図７は、Ｖｐｒ５２〜９６がミトコンドリアに結合することに対するＢｃｌ−２およびＰＡ−１０の差のある作用を表す。
Ｂ．Ｂｃｌ−２でなく、ＰＡ−１０が、ミトコンドリアに対するＶｐｒ５２〜９６の結合を阻害する。ミトコンドリアを示された阻害剤と１０分間プレインキュベーションして、ＦＩＴＣ−Ｖｐｒ５２〜９６で標識されたミトコンドリアの割合がＡのように測定される。Ｃ．ビオチン化Ｖｐｒ５２〜９６によるＡＮＴのアフィニティー精製に対するＢｃｌ−２の阻害作用。ミトコンドリアを、示された阻害剤とインキュベーションし、その後、５μＭのビオチン化Ｖｐｒ５２〜９６とともにＲＴで３０分間さらした。ミトコンドリアを、材料および方法に記載されるようにＴｒｉｓ／ＨＣｌとともに、ビオチン化Ｖｐｒ５２〜９６とのインキュベーション後（上段）またはインキュベーション前（下段）に溶解した。そのミトコンドリアリガンドとの複合体を形成したビオチン化Ｖｐｒ５２〜９６がアビジン−アガロースに保持され、ＡＮＴの免疫ブロット検出に供された。
【図７Ｃ】
図７は、Ｖｐｒ５２〜９６がミトコンドリアに結合することに対するＢｃｌ−２およびＰＡ−１０の差のある作用を表す。
【図８Ａ】
図８は、Ｖｐｒ−ＡＮＴ相互作用のＢｃｌ−２媒介による阻害を表す。
Ａ．Ｖｐｒ５２〜９６と精製された天然ＡＮＴとの相互作用のＢｃｌ−２媒介による阻害のプラスモン表面共鳴測定。相互作用は、示された濃度の組換えＢｃｌ−２、Ｂｃｌ−２Δα_{５ ／ ６}または組換えＢｉｄを添加した後に測定された。データ（Ｘ±ＳＤ、ｎ＝３）は図１のように計算された。
【図８Ｂ】
図８は、Ｖｐｒ−ＡＮＴ相互作用のＢｃｌ−２媒介による阻害を表す。
Ｂ．ＶｐｒがＡＮＴプロテオリポソームに結合することに対するＢｃｌ−２の作用。８００ｎＭのＢｃｌ−２または２μＭのＰＡ１０とプレインキュベーションされたＡＮＴプロテオリポソームにおけるＦＩＴＣ標識Ｖｐｒ５２〜９６の保持（Ｘ±ＳＤ、ｎ＝３）が図２Ａのように評価された。
【図８Ｃ】
図８は、Ｖｐｒ−ＡＮＴ相互作用のＢｃｌ−２媒介による阻害を表す。
Ｃ．平面状脂質二重層におけるＶｐｒ−ＡＮＴチャンネルの形成に対するＢｃｌ−２の作用。Ｖｐｒ５２〜９６＋ＡＮＴ＋Ｂａｘ（０．４：１：０．３ｎＭ）およびＶｐｒ５２〜９６＋ＡＮＴ＋Ｂｃｌ−２（０．４：１：１ｎＭ）の単一チャンネル記録（＋７５ｍＶ）ならびに対応する振幅ヒストグラムが示される。Ｖｐｒ５２〜９６＋ＡＮＴ単独に対する対照値は図１ｅ（示されず）の場合と同様である（ｃ、閉鎖、ｏ、開放）。
【図９】
図９はＶｐｒ／ＰＴＰＣ相互作用のモデルを表す。ＶｐｒはＶＤＡＣを通って外膜を横断するが、これは、Ｋｏｅｎｉｇｓポリアニンによって阻害される。Ｖｐｒは、その後、ＡＮＴと相互作用する。Ｂｃｌ−２およびＡＮＴリガンド（ボンクレキン酸塩）は、ＶｐｒがＡＮＴに結合することを阻害し、これに対して、ＣｓＡは、シクロフィリンＤ（Ｃｙｐ−Ｄ）に対するその作用を介してＡＮＴの細孔形成機能に間接的に影響を及ぼす。[0001]
This application claims the benefit of US Provisional Patent Application No. 60 / 231,539 (filed September 11, 2000) and US Provisional Patent Application No. 60 / 232,841 (filed September 15, 2000). , Both of which are incorporated herein by reference.
[0002]
(Field of the Invention)
The present invention relates to the proto-apoptotic protein Vpr encoded by HIV-1 whose physical association with the mitochondrial inner membrane protein ANT (adenine nucleotide transporter, also called adenine nucleotide translocase or ADP / ATP carrier). And the discovery that functional interactions induce mitochondrial membrane permeabilization. HIV-1 viral protein R (Vpr) interacts with the permeable transition pore complex (PTPC) to form ANT pore formation and / or mitochondrial membrane permeabilization (MMP) and consequent cell death (MMP). Cell death by apoptosis, or by some related mechanism of cell death).
[0003]
(Background of the Invention)
It is now recognized that mitochondria play an important role in controlling cell survival and death (apoptosis) (Kroemer and Reed, 2000). Thus, an increasing number of molecules are involved in signal transduction, and many metabolites (and certain viral effectors) appear to act on mitochondria and affect permeabilization of mitochondrial membranes. Also, a significant number of experimental anti-cancer drugs kill cells by acting directly on the mitochondrial membrane (Ravagnan et al., 1989; Larochette et al., 1999; Marchetti et al., 1999; Fulda et al., 1999; Belzacq et al., 2000). Thus, the use of specific pro-apoptotic agents for mitochondria appears to be a new concept in anti-cancer chemotherapy (for reference, Costantini et al., 2000). A possible outcome could be to use cytoprotective molecules based on their ability to stabilize mitochondrial membranes to treat diseases associated with excessive apoptosis (AIDS, neurodegenerative diseases, etc.). Against this background, the identification of such molecular components (modes of action) that control the permeability of mitochondrial membranes has become a major theme in biomedicine.
[0004]
MMPs release caspase activator and caspase-independent death effectors from the transmembrane space, median transmembrane potential (ΔΨ_m) As well as apoptotic cell death with disrupted oxidative phosphorylation. G. FIG. Kroemer, N .; Zamzami, S.M. A. Susin, Immunol. Today, 18, 44-51 (1997); R. Green, J.M. C. Reed, Science, 281, 1309-1312 (1998); J. Lemasters et al., Biochim. Biophys. Acta, 1366, 177-196 (1998); C. Wallace, Science, 283, 1482-1488 (1999); G. FIG. Vander Heiden, C.I. B. Thompson, Nat. Cell Biol. 1, E209-E216 (1999); Gross, J.M. M. McDonnell, S.M. J. Korsmeyer, Genes Dev. 13, 1988-1911 (1999); Kroemer, J .; C. Reed, Nat. Med. 6, 513-519 (2000). Pro-apoptotic and anti-apoptotic members of the Bcl-2 family interact with adenine nucleotide transporters (ANT; at the inner membrane (IM)), voltage-dependent anion channels (VDAC; at the outer membrane (OM)). And / or regulates inner and outer MMPs by autonomous channel-forming activity. G. FIG. Kroemer, N .; Zamzami, S.M. A. Susin, Immunol. Today, 18, 44-51 (1997); R. Green, J.M. C. Reed, Science, 281, 1309-1312 (1998); J. Lemasters et al., Biochim. Biophys. Acta, 1366, 177-196 (1998); C. Wallace, Science, 283, 1482-1488 (1999); G. FIG. Vander Heiden, C.I. B. Thompson, Nat. Cell Biol. 1, E209-E216 (1999); Gross, J.M. M. McDonnell, S.M. J. Korsmeyer, Genes Dev. 13, 1988-1911 (1999); Kroemer, J .; C. Reed, Nat. Med. 6, 513-519 (2000); Marzo et al., Science, 281, 2027-2031 (1998); Shimizu, M .; Narita, Y .; Tsujimoto, Nature, 399, 483-487 (1999); Shimizu, A .; Konishi, T .; Kodama, Y .; Tsujimoto, Proc. Natl. Acad. Sci. USA, 97, 3100-3105 (2000); Desagher et al. Cell Biol. 144, 891-901 (1999).
[0005]
ANT and VDAC are the major components of the permeable transition pore complex (PTPC), a polyprotein structure organized at the site where two mitochondrial membranes juxtapose. G. FIG. Kroemer, N .; Zamzami, S.M. A. Susin, Immunol. Today, 18, 44-51 (1997); R. Green, J.M. C. Reed, Science, 281, 1309-1312 (1998); J. Lemasters et al., Biochim. Biophys. Acta, 1366, 177-196 (1998); C. Wallace, Science, 283, 1482-1488 (1999); G. FIG. Vander Heiden, C.I. B. Thompson, Nat. Cell Biol. 1, E209-E216 (1999); Gross, J.M. M. McDonnell, S.M. J. Korsmeyer, Genes Dev. 13, 1988-1911 (1999); Kroemer, J .; C. Reed, Nat. Med. 6, 513-519 (2000); Crompton, Biochem. J. 341, 238-249 (1999).
[0006]
Adenine nucleotide transporters (ANTs) play an important role in processes that cause mitochondrial membrane permeabilization and consequent apoptosis (Marzo et al., 1998; Brenner et al., 2000). In the cellular environment, ANT is inserted into the inner mitochondrial membrane and has two opposing functions. On the one hand, ANT is an essential antiport for cellular bioenergetics and is specific for ATP and ADP. On the other hand, ANT can form non-specific lethal pores by the action of some ligands (natural or exogenous) that remove the mitochondrial electrochemical gradient.
[0007]
The HIV-1 regulatory protein Vpr has pleiotropic effects on viral replication and cell proliferation, differentiation, cytokine production, and NF-κB mediated transcription. M. Emerman, M .; H. Malim, Science, 280, 1880-1884 (1998); D. Frankel, J.M. A. T. Young, Annu. Rev .. Biochem. 67, 1-25 (1998); Bukrinsky, A .; Adzhubei, J .; Med. Virol. 9, 39-49 (1999). In addition, Vpr may be localized to mitochondria. I. G. FIG. Macleadie et al., Proc. Natl. Acad. Sci. USA, 92, 2770-2774 (1995); G. FIG. Macleadie et al., FEBS Lett. 410, 145-149 (1997); Mutami, L .; J. Montaner, V.A. Ayavoo, D.A. B. E. Weine, DNA and Cell Biology, 19, 179-188 (2000); Jacottot et al. Exp. Med. 191, 33-45 (2000). The full-length form of Vpr (Vpr1-96) or the synthesized truncated form acts on PTPC to produce ΔΨ_m, And all mitochondrial features of apoptosis, including the release of cytochrome c and apoptosis-inducing factor (AIF). E. FIG. Jacottot et al. Exp. Med. 191, 33-45 (2000). Vpr's MMP-inducing activity indicates that, in its C-terminal component (Vpr52-96), several key arginine (R) residues (R73, R77, R77, R73) are strongly conserved in various pathogenic HIV-1 isolates. R80) within an α-helix motif consisting of 12 amino acids (Vpr71-82). L. G. FIG. Macleadie et al., Proc. Natl. Acad. Sci. USA, 92, 2770-2774 (1995); G. FIG. Macleadie et al., FEBS Lett. 410, 145-149 (1997); Jacottot et al. Exp. Med. 191, 33-45 (2000).
[0008]
Depending on the apoptotic stimulus, permeabilization may affect OM and IM in an inconsistent manner, but permeabilization may or may not be accompanied by matrix swelling . G. FIG. Kroemer, N .; Zamzami, S.M. A. Susin, Immunol. Today, 18, 44-51 (1997); R. Green, J.M. C. Reed, Science, 281, 1309-1312 (1998); J. Lemasters et al., Biochim. Biophys. Acta, 1366, 177-196 (1998); C. Wallace, Science, 283, 1482-1488 (1999); G. FIG. Vander Heiden, C.I. B. Thompson, Nat. Cell Biol. 1, E209-E216 (1999); Gross, J.M. M. McDonnell, S.M. J. Korsmeyer, Genes Dev. 13, 1988-1911 (1999); Kroemer, J .; C. Reed, Nat. Med. 6, 513-519 (2000). In vitro experiments performed on purified mitochondria or purified proteins reconstituted into artificial membranes suggest at least two competing models of MMP. On the one hand, pore formation by the ANT results in permeation of the IM, swelling of the matrix by osmotic pressure, and consequent rupture of the OM (because the surface area of the IM with its folded crystallites exceeds that of the OM) Is caused to explain). In support of this hypothesis, Bax, atractyloside, Ca^{2 +}And pro-apoptotic molecules such as thiol oxidants cause ANT (which is usually a very specific ADP / ATP antiporter) to form non-specific pores (I. Marzo et al., Science, 281, 2027-2031 (1998); N. Brustovetsky, M. Klingenberg, Biochemistry, 35, 8483-8488 (1996); C. Brenner et al., Oncogene, 19, 329-336 (2000)). On the other hand, VDAC has been suggested to account for initial OM permeation without affecting IM. S. Shimizu, M .; Narita, Y .; Tsujimoto, Nature, 399, 483-487 (1999); Shimizu, A .; Konishi, T .; Kodama, Y .; Tsujimoto, Proc. Natl. Acad. Sci. USA, 97, 3100-3105 (2000). In support of this hypothesis, the permeabilization of VDAC-containing liposomes for sucrose or cytochrome c is enhanced by Bax and inhibited by Bcl-2 in vitro. S. Shimizu, M .; Narita, Y .; Tsujimoto, Nature, 399, 483-487 (1999); Shimizu, A .; Konishi, T .; Kodama, Y .; Tsujimoto, Proc. Natl. Acad. Sci. USA, 97, 3100-3105 (2000).
[0009]
Recent studies have revealed the presence of several viral apoptosis inhibitors that act on mitochondria. For example, adenovirus, Epstein-Barr virus, herpes virus (sairimi) and Kaposi's sarcoma-associated human herpes virus 8 produce Bcl-2 homologs that can suppress apoptosis. E. FIG. H. -Y. Cheng et al., Proc. Natl. Acad. Sci. USA, 94, 690-694 (1997); H. Han, D .; Modha, E .; White, Oncogene, 17, 2993-3005 (1998); Derfuss et al. Virol. 72, 5897-5904 (1998); L. Marshall et al. Virol. 73, 5181-5185 (1999). In addition, some viruses encode PTPC interacting proteins with no apparent homology to the Bcl-2 / Bax family. Cytomegalovirus apoptosis inhibitors pUL37x (VS Goldmacher et al., Proc. Natl. Acad. Sci. USA, 96, 12536-12541 (1999)) and Vpr (an apoptosis-inducing factor encoded by HIV-1) Selectively binds to ANT. The pro-apoptotic p13 (II) protein from the X-II ORF of HTLV-1 is also targeted to mitochondria via a peptide motif with structural similarity to the mitochondrial toxicity domain of Vpr. V. Ciminale et al., Oncogene, 18, 4505-4514 (1999). In addition, the pro-apoptotic MMP-induced hepatitis B virus protein X interacts with VDAC. Z. Rahmani, K .; W. Huh, R .; Lasher, A .; Siddiqui, J .; Virol. 74, 2840-2846 (2000). Thus, both VDACs and ANTs are key targets for viral apoptosis regulation, and possibly against viral etiologies and / or other pathologies associated with apoptotic dysregulation (ie, cancer, ischemia, neurodegenerative diseases, etc.). Revealed as a target for pharmacological intervention. Apoptosis is a process that appears in several phases: (1) the onset phase, which is very heterogeneous, during which biochemical pathways involved in the process are determined by apoptosis-inducing agents; ( 2) a determinant phase, which is common to various types of apoptosis, during which cells "decide" to commit suicide; and (3) a common degradation phase, which is a catabolic hydrolase (Caspase and nuclease) activation. Activation of caspases (cysteine proteases that cleave at aspartic acid [Asp] residues) and nucleases is necessary for the acquisition of complete apoptotic morphology, but inhibition of such enzymes is dependent on a number of different initiators. Cell death induced by (Bax, Bak, c-Myo, PML, FADD, glucocorticoid receptor occupancy, tumor necrosis factor, growth factor withdrawal, CXCR4 crosslinker, and chemotherapeutic agents (such as etoposide, camptothecin or cisplatin)) It seems clear that they do not inhibit. If caspases are not activated, the cells will show delayed cell lysis with no features of advanced apoptosis, such as global chromatin condensation, DNA fragmentation of oligonucleosomes, and formation of apoptotic bodies. However, prior to cell lysis, the cells were allowed to reach the inner transmembrane potential (Δ_m2) and / or permeabilization of both mitochondrial membranes with the release of apoptosis-producing proteins (such as cytochrome c and apoptosis-inducing factor (AIF)) through the outer membrane. These results invalidate the hypothesis that caspase activation is always required for apoptotic cell death to occur. Rather, cell death is closely linked to mitochondrial membrane permeabilization.
[0010]
Understanding apoptosis has recently been facilitated by the development of cell-free systems. Instead of considering the cell as a black box, the various subcellular fractions (eg, mitochondria, nucleus and cytoplasm) come together in order to reconstitute the apoptotic phenomenon by repeating the essential steps of the process in vitro. Mixed. It appears that proapoptotic second messengers, whose properties depend on apoptosis-inducing agents, accumulate in the cytoplasm during the onset phase. These factors then induce permeabilization of the mitochondrial membrane, thereby allowing cells to enter the determinant phase. Apoptotic changes in mitochondria are ΔΨ_mLoss, temporary swelling of the mitochondrial matrix, mechanical rupture of the outer membrane by permanent pores of large proteins and / or its non-specific permeabilization, and soluble transmembrane proteins (SIMP) that pass through the outer membrane ) In the release. Loss of mitochondrial membrane barrier function contributes to cell death by several factors, such as metabolic consequences at the bioenergetic level, loss of redox homeostasis, and disruption of ionic homeostasis. Activation of proteases (caspases) and nucleases by SIMP is required for the acquisition of apoptotic morphology. This latter phase corresponds to a degradation step beyond the point at which the apoptotic process does not reverse. Various SIMPs provide a molecular link between mitochondrial membrane permeabilization and catabolic hydrolase activation: (cytochrome c (a heme protein involved in caspase activation), certain procaspases (especially procaspases). 2 and 9, which are selectively concentrated in mitochondria in some cell types), and AIF). AIF is a nuclear-encoded transmembrane flavoprotein in which caspase-independent peripheral chromatin condensation and degradation of DNA into 50 kilobase pair fragments translocates to the nucleus induced by AIF.
[0011]
The mechanism of permeabilization of mitochondrial membranes is not completely understood. Some investigators report that the pro-apoptotic members of the Bcl-2 family oligomerize in an autonomous manner, ie, without requiring interaction with other mitochondrial membrane proteins, resulting in a persistent cytochrome c He prefers the hypothesis that such members are inserted into the outer membrane, which forms a fine pore. However, Bax-induced membrane permeabilization is inhibited by two inhibitors of the formation of permeable transition pores (or "megachannels"), cyclosporin A (CsA) and boncrekinic acid (BA). This suggests that sticky mitochondrial proteins (CsA and BA targets) are involved in this process. The permeable transition pore has a polyprotein structure formed at the site of contact between the inner and outer membranes. One of the important proteins of the permeable transition pore complex (PTPC) is the adenine nucleotide transporter (ANT). ANT is a target for BA, but is the most abundant inner membrane protein. ANT usually functions as a specific carrier protein for the exchange of adenosine triphosphate (ATP) and adenosine diphosphate (ADP), but can become non-specific pores.
[0012]
An interesting property of PTPC is that permeabilization of the inner and / or outer mitochondrial membrane impairs the bioenergetic equilibrium of cells (for example, PTPC reduces reduced NADPH and glutathione, oxidizes ATP, and reduces ATP). , ΔΨ_mAnd (eg, from the matrix Ca^{2 +}) To bring about intracellular ion homeostasis). Interestingly, all of these changes themselves increase the likelihood of PTPC opening. This has two important implications. First, the consequence of PTPC opening itself is in favor of PTPC opening in a self-amplifying loop that regulates lethal responses between mitochondria in the same cell. Secondly, this means that the end result that the PTPC opens is a global effect that is stylized by various biochemical changes, the effect of which is the specific pro-apoptotic signal transduction. Even if it is a cascade or non-specific damage at the energy or redox levels, it means not dependent on an initiating stimulus.
[0013]
Chemotherapy aims to specifically eradicate cancer cells, mainly by inducing apoptosis. In gene therapy, a Bax delivery vector can be used, which allows indirect targeting of mitochondria to induce apoptosis. In contrast to such proteins, certain peptides can easily penetrate the plasma membrane and thus can be used as true pharmacological agents. Mastoparan (a peptide isolated from bee venom) induces mitochondrial membrane permeabilization through a mechanism that can be inhibited by CsA, and when added to intact cells, induces apoptosis through the action of mitochondria. It is the first peptide known to induce. This peptide has an α-helical structure and has several positive charges distributed on one side of the helix. Similar peptides (KLAKLAKKLAKLAK, ie (KLAKLAK)₂(K = lysine, L = amine, and A = leucine)) were recently found to disrupt mitochondrial membranes when added to purified mitochondria, but the mechanism of action has not yet been elucidated. (Ellerby, HM et al., Anticancer Activity of Targeted Proapoptotic Peptides, Nature Med. 5, 1032-1038 (1999)).
[0014]
The viral protein R (Vpr), a pro-apoptotic 96 amino acid protein derived from human immunodeficiency virus-1, has a similar structural motif (aa 71-82), ie, several cations concentrated on the same side of the helix. Includes charged alpha helix. Vpr, as well as Vpr derivatives containing this “mitochondrial toxicity” domain, are ΔΨ_mCauses rapid elimination, inhibited by CsA and BA, and mitochondrial release of apoptogenic proteins such as cytochrome c or AIF. The same structural motif associated with killing cells is thought to be responsible for the mitochondrial toxic effects of Vpr. Vpr favors the permeabilization of an artificial membrane containing purified PTPC or a defined PTPC component such as ANT combined with Bax, but the effect of recombinant Bcl Inhibited by the addition of -2. According to surface plasmon resonance studies, the C-terminus of Vpr binds purified ANT with high affinity in the nanomolar range. E. FIG. Jacottot et al. Exp. Med. 191, 33-45 (2000): which is specifically incorporated herein by reference. Furthermore, the biotinylated Vpr-derived peptide (Vpr52 to 96) can be used as a bait for specifically purifying a mitochondrial molecular complex containing ANT and VDAC. Yeast strains without ANT or VDAC are less susceptible to Vpr-induced killing than control cells. Thus, Vpr induces apoptosis via a direct effect on mitochondrial PTPC. Like Vpr, the p13 (II) protein from the HTLV-1 X-II open reading frame is targeted to mitochondria, and ΔΨ_mLoss and mitochondrial swelling. Mitochondrial targeting of this protein has been mapped to a decapeptide sequence containing several Arg residues that are asymmetrically distributed within the alpha helix. However, Arg-Ala substitutions in the mitochondrial toxicity domain of p13 (II) do not abrogate mitochondrial targeting of p13.
[0015]
The lethal peptide can be targeted to mitochondria, and more specifically to PTPC, at least in the case of Vpr. Have recently reported that mitochondrial toxicity (KLAKLAK)₂The motif was fused to a targeting peptide that interacts with endothelial cells. Such a fusion peptide is internalized and induces permeabilization of mitochondrial membranes in angiogenic endothelial cells and kills MDA-MD-435 breast cancer xenografts implanted in nude mice. Similarly, a recombinant chimeric protein containing an interleukin-2 (IL-2) protein fused to Bax selectively binds to and kills IL-2 receptor-bearing cells in vitro. Thus, specific cytotoxic agents that target surface receptors are transported into the cytoplasm, induce apoptosis by mitochondrial membrane permeabilization, and may be useful in treating cancer.
[0016]
The problem of relapse with conventional chemotherapeutic agents is that chemotherapeutic agents can be impaired by alterations such as mutations in p53, increased antioxidant activity, blockade of the CD95 / CD95L pathway, overexpression of Bcl-2-like proteins. Is to utilize the apoptosis induction pathway. One of the possible ways to cause cell death is to trigger events downstream of a common apoptotic pathway. Thus, adenovirus-mediated transfer of caspases has been proposed as one method to induce cell death beyond any regulation. An alternative method is to use a mitochondrial toxic agent that induces cell death regardless of the upstream regulatory mechanism and regardless of the status of caspases and endogenous caspase inhibitors. As an example, LND, arsenite or CD437 induces cell death independent of p53 status by pathways unaffected by caspase inhibitors. Similarly, betulinic acid and Vpr cause apoptosis independent of CD95 (Apo-1 / Fas) and p53, and both increase the permeability of mitochondrial membranes in a caspase-independent manner. As a result, these types of agents may prove to be very useful in killing normally resistant cells. Moreover, the future of tumor therapy will benefit from the design of drugs that overcome Bcl-2-mediated stabilization of mitochondrial membranes, and from targeting amphipathic peptides or peptidomimetics to defined cell populations or tissues. You might get it.
[0017]
Selective eradication of transformed cells by use of mitochondrial-specific agents should be effective. One method is to target toxic agents to selected cell types based on the specific expression of the surface receptor. Another method to be developed in the future aims to exploit the differences in the composition or regulation of PTPC between normal and tumor cells. Future studies indicate that cell targeting (by use of retroviral or adenovirus vectors, use of integrin-specific domains, etc.) and / or tumor-specific alterations in PTPC may be in cancer therapy and also in mitochondrial Treatment of neurodegenerative diseases thought to be related to dysfunction of Friedreich's ataxia, hereditary spastic paraplegia, Huntington's disease, amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease, and levels of MMP (Kroemer, G. et al., Mitochondrial control of cell death, Nature Med. 6, No. 5, 513-519 (1999)).
[0018]
Thus, there is a need in the art for methods and reagents for regulating mitochondrial permeabilization and apoptosis.
[0019]
(Summary of the Invention)
The present invention relates to the physical and functional interaction of Vpr with the adenine nucleotide transporter (ANT), which functions to increase the permeability of mitochondrial membrane and cause cell death by apoptosis. In a preferred embodiment, the present invention relates to the physical and functional interaction of Vpr with three human isotypes of ANT, which are also called ANT1, ANT2 and ANT3. The present invention encompasses methods that utilize this novel mechanism to increase the permeability of mitochondrial membranes. The invention further includes a method of causing cell death by apoptosis.
[0020]
The invention also includes a method of altering or preventing Vpr from binding to ANT. The invention further includes a method of altering or preventing channel formation by associating Vpr with ANT. The invention also includes a method of causing or preventing mitochondrial membrane permeation. The invention also includes a method of causing or preventing apoptotic cell death.
[0021]
The invention also includes a method of screening for a molecule that alters or prevents Vpr from binding to ANT. The invention further includes a method of screening for molecules that alter or prevent channel formation by the association of Vpr with ANT. The invention also includes a method of screening for molecules that cause or prevent mitochondrial membrane permeation. The invention also includes a method of screening for a molecule that causes or prevents apoptotic cell death.
[0022]
The invention also includes a method of screening for molecules that compete with the binding of the C-terminal component of Vpr (vpr 52-96 for HIV-1) to ANT. The invention also includes a method of screening for molecules that promote the binding of the C-terminal component of Vpr (vpr 52-96 for HIV-1) to ANT. The invention also encompasses methods of screening for molecules that alter or prevent the binding of the C-terminal component of Vpr (vpr 52-96 for HIV-1) to ANT. The invention further encompasses methods of screening for molecules that alter or prevent mitochondrial membrane permeabilization by associating the C-terminal component of Vpr (vpr 52-96 for HIV-1) with ANT. The invention further encompasses methods of screening for molecules that alter or prevent apoptosis by associating the C-terminal component of Vpr (vpr 52-96 for HIV-1) with ANT.
[0023]
The invention also includes peptidic or non-peptidic molecules that alter or prevent Vpr from binding to ANT. The present invention also includes peptidic or non-peptidic molecules that mimic Vpr or Vpr fragments in their ability to physically or functionally interact with ANT. The invention further includes peptidic or non-peptidic molecules that alter or prevent channel formation by the association of Vpr with ANT. The invention also includes peptidic or non-peptidic molecules that cause or prevent mitochondrial membrane permeation. The invention also includes peptidic or non-peptidic molecules that cause or prevent apoptotic cell death. The invention further encompasses pharmaceutical and diagnostic compositions comprising these molecules, and the use of these compositions to cause or prevent mitochondrial membrane permeation or apoptosis.
[0024]
The invention further includes peptidic or non-peptidic molecules that mimic the C-terminal component of Vpr (vpr 52-96 for HIV-1) and modulate mitochondrial membrane permeabilization. The invention also includes peptidic or non-peptidic molecules that compete with the binding of the C-terminal component of Vpr (vpr 52-96 for HIV-1) to ANT. The invention also includes peptidic or non-peptidic molecules that facilitate the binding of the C-terminal component of Vpr (vpr 52-96 for HIV-1) to ANT. The invention further encompasses pharmaceutical and diagnostic compositions comprising these molecules, and the use of these compositions to cause or prevent mitochondrial membrane permeation or apoptosis.
[0025]
The invention also includes a method of screening for genetic or epigenetic changes in the expression or structure of three ANT isotypes in humans. The invention further encompasses screening and diagnosis for differences in the ability of three ANT isotypes to interact with Vpr in various patients to promote mitochondrial membrane permeation, channel formation and / or apoptosis.
[0026]
The invention also includes a method for specifically killing cells by inducing apoptosis.
[0027]
The invention also includes a method for screening for a molecule that alters the channel properties of an ANT.
[0028]
The invention further includes a method of screening for active molecules that can alter or prevent ANT-Bcl2 interaction.
[0029]
By studying the cytotoxicity of the HIV-1 Vpr protein, we have discovered that Vpr interacts directly with ANT to cause mitochondrial membrane permeabilization as well as apoptosis. First, Vpr crosses the outer mitochondrial membrane using mitochondrial proteins (also called "voltage-gated anion channels": VDACs), and then passes itself to its primary target, ANT. , With strong affinity (KD = 1 nM). The ANT / Vpr complex is responsible for the permeabilization of the inner mitochondrial membrane, the swelling of the mitochondrial matrix, and ultimately the destruction of the outer membrane, and thus various factors (eg, AIF, cytochrome c and some Procaspase) to form a high conductance channel that triggers the release. We have identified an interaction site between ANT and Vpr for Vpr (14 Kd; 96 aa): the binding site for ANT includes a pattern of 71HFFRIGCRHSRIG82 (minimal toxicity pattern) of Vpr52-96 (amino acids 52-96). It is utilized in the center of an α-helical (between ８３83) linear structure. In the case of ANT1 (30 kd; 298 aa), the pattern of 104DRHKQFWRYFAGN116 is used in the binding site for Vpr in the center of the second ANT ring (aa92-116).
[0030]
With the discovery of at least one of the physical and functional interactions between Vpr and ANT and the site of interaction, the inventors have discovered that ANT-containing protein complexes (permeability transition cells). Pore; PTPC), which has led to the assembly of analogs of this toxicity pattern of Vpr. These molecules may serve to mimic the pro-apoptotic effects of Vpr to destroy cancerous cells in vitro or in vivo. These molecules are either peptide or non-peptide molecules and are obtained in isolated or purified form.
[0031]
The present invention relates to a novel protein / protein interaction between the retroviral HIV regulatory protein Vpr and mitochondrial adenine nucleotide transporter (ANT), a membrane-bound receptor involved in the control of cell death by apoptosis. The invention also relates to peptidic or non-peptidic molecules that have the ability to alter and / or prevent Vpr from binding to ANT (or channel formation by this binding). Another aspect of the invention is the C-terminal component of Vpr (Vpr52 for HIV-1) in its ability to bind and cooperate with ANT to increase the permeability of mitochondrial membranes (and thus kill cells). ~ 96), which has the ability to mimic peptidic or non-peptidic molecules. The invention also relates to pharmaceutical compositions containing an effective amount of a molecule that alters and / or prevents Vpr from binding to ANT (and / or the conformational consequences of this binding, such as channel formation) and It relates to a diagnostic composition (therefore such a composition is cytoprotective), and also to a therapeutic or diagnostic method using such a pharmaceutical or diagnostic composition. In addition, the present invention also relates to molecules that can bind to ANT and mimic the C-terminal component of Vpr in its ability to increase the permeability of mitochondrial membranes (and thus kill cells) in cooperation with ANT. It relates to pharmaceutical and diagnostic compositions containing an effective amount, and also to therapeutic or diagnostic methods using such pharmaceutical or diagnostic compositions. The present invention also relates to the ability to alter and / or prevent Vpr from binding to ANT (or channel formation due to this binding), or to bind ANT, and cooperate with ANT in mitochondrial membrane permeability. To screen for new active molecules (endogenous or exogenous) that have the ability to mimic the C-terminal component of Vpr (Vpr52-96 for HIV-1) in their ability to increase (and thus kill cells) Related to Finally, the invention relates to methods of screening for genetic or epigenetic changes in the expression or structure of three ANT isotypes in humans, such as specific changes in individuals affected by cancer.
[0032]
Thus, the present invention relates to protein-protein interactions between Vpr and ANT, and potentially protein-protein interactions between Vpr and VDAC. This screens for therapeutic molecules that are active as cytoprotective factors (inhibitors of the ANT / Vpr interaction) or that are cytotoxic (active analogs of Vpr for interaction with ANT and / or VDAC). Can be used for In this regard, we have established a new ELISA screening test for Vpr ligands and molecules that can inhibit the binding of ANT to Vpr.
[0033]
Therefore, one of the objects of the present invention is to provide a peptide molecule or a peptide molecule capable of mimicking Vpr by binding to ANT in cells (or specific cells) of an individual (specifically, a person affected by cancer). For non-peptide molecules.
[0034]
The present invention also provides structural or functional inhibitors that are effective in blocking Vpr / ANT interactions or Vpr / VDAC interactions, thus: 1) Inhibiting HIV infection in vitro or in vivo. Structural or functional inhibitors, and 2) inhibit the cytotoxic effects of any ANT ligand (natural, endogenous, exogenous) and therefore protect cells in patients affected by diseases associated with excessive apoptosis It relates to a structural or functional inhibitor that produces an effect.
[0035]
Thus, the invention also includes, on the one hand, Vpr (which is found in the intracellular, extracellular fluid, or HIV particles of retrovirally infected individuals) or analogs of Vpr (endogenous [eg, Bcl-2 or its Or exogenous) and, on the other hand, components that can alter the interaction with at least one of the isotypes of ANT found in cells at the mitochondrial membrane level. Molecules derived from ANT or from patterns of interaction with Vpr (eg, ANT 104-116) are also considered active molecules that form part of the present invention.
[0036]
The invention also relates to the use of the compounds and inhibitors defined earlier as active ingredients of pharmaceutical compounds. One possible specific application is to screen tumors in vivo for Vpr derived from a Vpr pattern (eg, a pattern of 52-96, 71-82 or 71-96) or derived from an analog of Vpr. It is thought that it is possible to couple with a molecule. Thus, the present invention encompasses the use of Vpr patterns (e.g., patterns 52-96, 71-82 or 71-96).
[0037]
The invention also includes means for screening for molecules that can mimic the cytotoxic and / or mitochondrial effects of Vpr (specifically, its interaction with ANT and / or VDAC), and On the one hand, Vpr (which is found in the intracellular, extracellular fluid, or HIV particles of retrovirally infected individuals) or analogs of Vpr (endogenous or exogenous) and, on the other hand, intracellularly at the mitochondrial membrane level. Means for screening for molecules (cytoprotective) capable of altering the interaction of the found ANT with at least one of the isotypes are included.
[0038]
Thus, the present invention provides for the selection of ANT agonists (structural or functional analogs of Vpr) or antagonists (inhibitors of the Vpr / ANT interaction, or "lethal pore" conformations responsive to Vpr; Or structural or functional analogs of Vpr). Thus, the present invention includes at least two screening tests:
-Binding test of ANT (or, for example, an ANT-derived peptide containing ANT patterns 104-116) to Vpr (or, for example, a Vpr-derived peptide containing patterns 71-82). The protocol of this test is already established when ANT or ANT peptide containing patterns 104-116 is bound and then the exposed Vpr 52-96 is bound to the bottom of a 96-well plate.
A test called a “double test” functional for ANT, which simultaneously evaluates the specific antiport function and the nonspecific lethal pore function of ANT. This test principle and detailed protocol are described in Example 5.
[0039]
(Description of Embodiment)
The present invention relates to the discovery that the pro-apoptotic protein Vpr encoded by HIV-1 has its physical and functional interaction with the inner mitochondrial membrane protein ANT (adenine nucleotide transporter, also called ADP / ATP carrier). Related to the discovery that it induces mitochondrial membrane permeation through action. It uses a variety of different techniques to study, for example, surface plasmon resonance, electrophysiology, synthetic proteoliposomes, purified mitochondria (respirometry, electron microscopy, organelle fluorescence measurements), and microscopic analysis of intact cells. Indicated using injection. The mode of action of Bcl-2 acts on ANT to prevent Vpr-mediated mitochondrial action.
[0040]
The present invention relates to the discovery that Vpr primarily affects the permeability of IM, but not OM, in vitro. Vpr binds to ANT in a ANT conformation-dependent manner (FIGS. 1 and 2) and cooperates with ANT to form a channel (FIG. 3) in which OM binds to cytochrome c. Increase the permeability of the IM before it becomes transparent (FIGS. 4 and 5). Based on two independent observations, Bcl-2 neutralizes this effect. First, its mode of action is distinctly different from that of the VDAC inhibitor PA10 (FIGS. 6 and 7). Second, Bcl-2 can influence physical and functional ANT-Vpr interactions in synthesized VDAC-free systems (FIG. 8). These data indicate that Bcl-2 and other members of the Bcl-1 family show relatively large globular proteins (linear predominantly α-helical structures of Vpr52-96 as revealed by NMR (W. Schuller et al., J. Am. Mol. Biol. 285, 2105-2117 (1999)), but does not exclude the possibility of modulating the permeability of VDAC to 14.5 kDa for cytochrome c, but at least in this particular model, ANT Shows that Bcl-2 exerts its membrane-protective mitochondrial action.
[0041]
HIV-1 viral protein R (Vpr) interacts with the permeable transition pore complex (PTPC) to cause mitochondrial membrane permeabilization (MMP) and consequent apoptosis. Vpr binds to the inner mitochondrial membrane protein, adenine nucleotide transporter (ANT). E. FIG. Jacottot et al. Exp. Med. 191, 33-45 (2000). When Vpr binds to ANT, they together form a large conductance channel in the composite membrane. When added to purified mitochondria, Vpr uncouples with the respiratory chain and induces a rapid inner MMP that precedes the outer MMP for cytochrome c, matrix swelling induced by Vpr, and inner for proton and NADH. MMPs are prevented by preincubating purified mitochondria with recombinant Bcl-2 protein. In contrast to Konigs polyanine, Bcl-2, a specific inhibitor of the voltage-gated anion channel (VDAC), cannot prevent Vpr from crossing the outer mitochondrial membrane. Bcl-2 reduces ANT-Vpr interaction and abolishes channel formation by the ANT-Vpr complex. Thus, both Vpr and Bcl-2 regulate MMPs by direct interaction with ANT.
[0042]
Methods of altering or preventing Vpr from binding to ANT
The discovery that Vpr interacts physically and functionally with ANT allows for ways to alter or prevent Vpr from binding to ANT. As exemplified in Examples 1-4, Vpr interaction with ANT can be detected and modulated with a variety of different assay systems. For example, Bcl-2 regulates the physical and functional interaction of Vpr with ANT. Similarly, ANT104-116 of the peptide corresponding to the overlap between the binding motif of Bcl-2 and the second ANT loop inhibit ANT-Vpr binding. Thus, these molecules can be used to alter or prevent Vpr from binding to ANT. Other peptidic or non-peptidic molecules can be designed to inhibit this binding as well.
[0043]
Identifying Vpr-ANT binding allows the generation of molecules that can regulate apoptosis. The methods set forth in the examples and other conventional techniques can be adapted to screen for Vpr, Bcl-2 or ANT variants, or other polypeptides or molecules that affect Vpr-ANT binding. . This allows for the production of molecules that can enhance or inhibit Vpr-ANT binding. The activity of these molecules can be assessed by competitive binding assays. For example, various molecules can be evaluated for their ability to inhibit ANT-Vpr binding using the binding assays described in the Examples. One skilled in the art will appreciate that many other techniques may be used as well. The identified molecules can be further evaluated for apoptotic activity by conventional techniques. In addition, based on the structure of Vpr molecules that have been shown to bind to ANT, structurally similar molecules can be designed to mimic or inhibit Vpr activity.
[0044]
In one embodiment, a soluble form of the Vpr or ANT polypeptide can be incubated with the cells to enhance or inhibit the induction of apoptosis. In one embodiment, these polypeptides contain a mutation that prevents apoptosis. In another embodiment, these polypeptides contain a mutation that enhances apoptosis. In one embodiment, these polypeptides are synthesized. In another embodiment, these polypeptides are produced by recombinant techniques.
[0045]
Vpr and ANT polypeptides that inhibit or enhance Vpr-ANT binding, mitochondrial membrane permeabilization or apoptosis and peptides with more than 9 amino acids have at least 10-20 amino acids, 20-30 amino acids, 30 It is an aspect of the present invention, as is a peptide of size 50 to 50 amino acids, 50 to 100 amino acids, and 100 to 365 amino acids. DNA fragments encoding these polypeptides and peptides are encompassed by the present invention.
[0046]
Synthetic polypeptides and peptides can be made by a variety of conventional techniques. Such techniques include: Merrifield, Methods Enzymol. 289: 3-13, 1997; Ball and P.M. See Mascagni, Int. J. Pept. Protein Res. 48: 31-47, 1996; See Molina et al., Pept. Res. 9: 151-155, 1996; Fox, Mol. Biotechnol. 3: 249-258, 1995; Lepage et al., Anal. Biochem. 213: 40-48, 1993.
[0047]
In another embodiment, the peptide can be prepared by subcloning a DNA sequence encoding the desired peptide sequence into an expression vector for producing the desired peptide. The DNA sequence encoding the peptide is conveniently linked to a sequence encoding a suitable leader or signal peptide. Alternatively, DNA fragments can be chemically synthesized using conventional techniques. DNA fragments may also be clones of DNA (eg, HIV-1) clones using known restriction enzymes (New England Biolabs (1997 catalog), Stratagene (1997 catalog), Promega (1997 catalog)). It can be made by restriction endonuclease digestion and isolated by conventional means, such as by agarose gel electrophoresis.
[0048]
In another embodiment, well known polymerase chain reaction (PCR) techniques can be used to isolate and amplify the DNA sequence encoding the desired protein fragment. Oligonucleotides that define the desired termini of the DNA fragment are used as 5 'and 3' primers. The oligonucleotide may include a recognition site for a restriction endonuclease to facilitate insertion of the amplified DNA fragment into an expression vector. PCR technology is described in Saiki et al., Science, 239: 487 (1988); Recombinant DNA Methology (Wu et al., Eds., Academic Press, Inc., San Diego, 1989), pp 189-196; Innis et al., Academic Press, Inc., 1990). Of course, it is understood that many techniques can be used to prepare polypeptide and DNA fragments, and that this embodiment in no way limits the scope of the invention.
[0049]
Screening method using Vpr and ANT
Vpr or ANT polypeptides can be evaluated for their ability to mediate apoptosis, as well as to inhibit Vpr-mediated apoptosis. For example, a fragment of Vpr can be evaluated for its ability to block the binding of native Vpr to ANT by conventional titration experiments.
[0050]
In one embodiment, surface plasmon resonance is used to assess Vpr binding to ANT as described herein. In another aspect, electrophysiology is used to assess Vpr binding to ANT as described herein. In another aspect, purified mitochondria are used to assess Vpr binding to ANT as described herein. In another aspect, the synthesized proteoliposomes are used to assess Vpr binding to ANT as described herein. In another aspect, microinjection of living cells is used to assess Vpr binding to ANT as described herein. Of course, it is understood that many techniques can be used to assess the binding of Vpr to ANT, and that these embodiments in no way limit the scope of the invention.
[0051]
In another embodiment, the yeast two-hybrid system developed at SUNY, which is disclosed in US Patent No. 5,283,173 (Fields et al.); J. Luban and S. Goff, Curr Opin. Biotechnol. R. Brachmann and J. Boeke, Curr Opin. Biotechnol. 8: 561-568, 1997; R. Brent and R. Finley, Ann. Rev. Genet. 31: 663-704, 1997; P. Bartel. And S. Fields, Methods Enzymol. 254: 241-263, 1995) can be used to screen for inhibitors of the Vpr-ANT interaction as described below. Vpr or its part involved in the interaction is fused to the DNA binding domain of Gal4 and, together with the ANT molecule fused to the Gal4 transcription activation domain, is introduced into a strain whose growth on histidine-free plates depends on Gal4 activity. be able to. The interaction of the Vpr polypeptide with an ANT molecule allows the growth of yeast containing both molecules, and screens for molecules that inhibit or alter this interaction (ie, inhibit or increase proliferation). Screening).
[0052]
In an alternative embodiment, a detectable marker (eg, β-galactosidase) can be used to measure binding in a yeast two-hybrid system.
[0053]
In addition, identifying Vpr as an ANT binding molecule allows for a method of detecting and quantifying ANT expression in cells. For example, the level of ANT can be revealed by contacting a labeled Vpr polypeptide with a biological sample containing ANT and detecting the Vpr-ANT complex.
[0054]
Purified ANT polypeptides, including proteins, polypeptides, fragments, variants, oligomers and other forms, are capable of binding Vpr in any suitable assay, including conventional binding assays. Can be tested for Similarly, Vpr polypeptides, including proteins, polypeptides, fragments, variants, oligomers and other forms, can be tested for their ability to bind ANT. To illustrate, Vpr polypeptides can be labeled with a detectable reagent (eg, a radionucleotide, a chromophore, an enzyme that catalyzes a colorimetric or fluorometric reaction, etc.). The labeled polypeptide is contacted with a cell expressing the ANT. The cells are then washed to remove unbound labeled polypeptide, and the presence of the label bound to the cells is determined by a suitable technique selected according to the nature of the label.
[0055]
Alternatively, the binding properties of Vpr polypeptides and polypeptide fragments can be determined by analyzing the binding of Vpr polypeptides and polypeptide fragments to ANT-expressing cells by FACS analysis and / or immunofluorescence. This allows one to characterize the binding of Vpr and ANT polypeptides and polypeptide fragments and to discriminate the relative ability of Vpr polypeptides and polypeptide fragments to bind ANT. In vitro binding assays using Vpr and ANT can also be used to characterize Vpr-ANT binding activity.
[0056]
Another type of suitable binding assay is a competitive binding assay. To illustrate, the biological activity of a variant can be measured by assaying for the ability of the variant to compete with the native protein for binding to Vpr or ANT.
[0057]
Various competitive binding assays can be performed by conventional methodologies. Reagents that can be used in competitive binding assays include radiolabeled Vpr, and intact cells expressing ANT (endogenous or recombinant). For example, radiolabeled Vpr fragments can be used to compete with soluble Vpr variants for binding to ANT in cells. Instead of intact cells, ANT protein bound to a solid phase can be substituted.
[0058]
Another type of competitive binding assay uses radiolabeled Vpr and isolated mitochondria. While quantitative results can be obtained by competitive plate binding assays by autoradiography, Scatchard plots (Scatchard, Ann. NY Acad. Sci. 51: 660, 1949) Can be used to get results.
[0059]
Peptidic or non-peptidic molecules that affect the interaction of Vpr on ANT or mimic its ability to interact with ANT
Mutant
The present invention includes variants of Vpr or ANT whose binding activity is altered. Variant polypeptides provided herein include variants of the native polypeptide that retain the natural biological activity, or substantial equivalents thereof. One example is a variant that binds with essentially the same binding affinity as the native form does. Binding affinity can be measured by conventional techniques, for example, as described in US Pat. No. 5,512,457, and as shown below.
[0060]
Variants can also bind with increased affinity. In one embodiment, the variant is an agonist of native Vpr on the biological activity of ANT. In another aspect, the variant is an antagonist of native Vpr on the biological activity of ANT. Agonist or antagonist activity can be readily measured by the techniques described herein.
[0061]
Variants include polypeptides that are substantially homologous to the native form, but have an amino acid sequence that differs from the native form by one or more deletions, insertions, or substitutions. Specific embodiments include, but are not limited to, polypeptides that contain from 1 to 10 amino acid residue deletions, insertions or substitutions when compared to the native sequence.
[0062]
Certain amino acids can be substituted, for example, with residues having similar physiochemical properties. Examples of such conservative substitutions include substitutions between aliphatic residues (mutations such as He, Val, Leu or Ala), polar residues (eg, between Lys and Arg, between Glu and Asp). Substitution, or between GIn and Asn), or between aromatic residues (such as Phe, Trp or Tyr). Other conservative substitutions are well known, for example, involving substitutions of entire regions having similar hydrophobic properties. Mutants can be made using conventional techniques, including random or site-directed mutagenesis.
[0063]
antibody
In aspects of the invention, Vpr and ANT polypeptides and peptides based on the amino acid sequences of Vpr and ANT can be used to prepare antibodies that specifically bind to Vpr and ANT polypeptides. . Provided herein are antibodies having immunoreactivity with a polynucleotide of the invention. In this aspect of the invention, polypeptides based on the amino acid sequences of Vpr and ANT can be used to prepare antibodies that specifically bind to Vpr and ANT. Such antibodies specifically bind to the polypeptide via the antigen-binding site of the antibody (as opposed to non-specific binding). Therefore, the polypeptides, fragments, mutants, fusion proteins and the like shown above can be used as immunogens for producing antibodies having immunoreactivity with them. More specifically, polypeptides, fragments, variants, fusion proteins and the like contain antigenic determinants or epitopes that trigger the formation of antibodies.
[0064]
These antigenic determinants or epitopes can be either linear or conformational (discontinuous). Linear epitopes are composed of a single portion of the amino acids of the polypeptide, whereas conformational or discontinuous epitopes are amino acids from various regions of the polypeptide chain that are in close proximity when the protein folds. (CA Janeway, Jr. and P. Travers, Immuno Biology, 3: 9 (Garland Publishing Inc., 2nd edition, 1996)). Since the folded protein has a complex surface, the number of epitopes available is quite large. However, due to the conformation and steric hindrance of the protein, the number of antibodies that actually bind to the epitope is less than the number of available epitopes (CA Janeway, Jr. and P. Travelers, Immuno). Biology, 2:14 (Garland Publishing Inc., 2nd edition, 1996)). Epitopes can be identified by any of the methods known in the art.
[0065]
Accordingly, one aspect of the present invention relates to an antigenic epitope of a polypeptide of the present invention. Such epitopes are useful for producing antibodies, particularly monoclonal antibodies, as described in detail below. In addition, epitopes derived from the polypeptides of the invention may be used as research reagents in assays and to purify specific binding antibodies from substances such as polyclonal sera or supernatants obtained from cultured hybridomas. Can be. Such epitopes or variants thereof are produced using techniques well known in the art, such as solid phase synthesis, chemical or enzymatic cleavage of the polypeptide, or using recombinant DNA technology. can do.
[0066]
For antibodies that can be elicited by an epitope of a polypeptide of the present invention, both polyclonal and monoclonal antibodies are described below, even if the epitope is isolated or remains part of the polypeptide. It can be prepared by such conventional techniques.
[0067]
The term "antibody" refers to polyclonal antibodies, monoclonal antibodies, fragments thereof, specifically antigen-binding fragments such as F (ab ') 2 fragments and Fab fragments, and any recombinantly produced binding partners. It is meant to include The antibody is about 10⁷M^-1Or higher or equivalent K_aIs defined as binding specifically when binding with. The affinity of the binding partner or antibody can be determined using conventional techniques, for example, as described in Scatchard et al., Ann. N. Y. Acad. Sci. 51: 660 (1949).
[0068]
Polyclonal antibodies are readily produced from a variety of sources, for example, from horses, cows, goats, sheep, dogs, chickens, rabbits, mice or rats, using techniques well known in the art. Can be. In general, purified Vpr (or ANT), which is appropriately conjugated, or a peptide based on the amino acid sequence of Vpr (or ANT), which is appropriately conjugated, is administered to a host animal, typically parenterally. Administered by injection. The immunogenicity of Vpr (or ANT) can be increased by the use of an adjuvant (eg, Freund's complete adjuvant or Freund's incomplete adjuvant). Following immunization with a boost, a small blood sample is taken and tested for reactivity to Vpr or ANT polypeptide. Examples of various assays useful for such measurements include the assays described in Antibodies: A Laboratory Manual (Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988); and countercurrent immunoelectrophoresis (CIEP). ), Radioimmunoassay, radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA), dot blot assays and sandwich assays. See U.S. Patent Nos. 4,376,110 and 4,486,530.
[0069]
Monoclonal antibodies can be readily prepared using a variety of well-known techniques. For example, U.S. Pat. No. Re. 32,011, U.S. Pat. Nos. 4,902,614, 4,543,439 and 4,411,993; Monoclonal Antibodies, Hybridomas: A New Dimension in Biological. Analysses, Plenum Press, Kennett, McKeam and Bechtol (eds.), 1980). Briefly, isolated and purified Vpr (or ANT), conjugated Vpr (or ANT) peptides are added to a host animal, such as a mouse, at least once, optionally in the presence of an adjuvant. Is injected intraperitoneally at least twice at approximately three week intervals. The mouse sera is then assayed by conventional dot blot techniques or antibody capture (ABC) to determine which animals are best for the fusion. Approximately two to three weeks later, the mice are given an intravenous boost of Vpr (or ANT) peptide or conjugated Vpr (or ANT) peptide. Thereafter, the mice are sacrificed and spleen cells are fused with commercially available myeloma cells such as Ag8.653 (ATCC) according to established protocols. Briefly, myeloma cells are washed several times with medium and fused to mouse spleen cells at a spleen cell to myeloma cell ratio of about 3: 1. The fusion agent can be any suitable agent used in the art (eg, polyethylene glycol (PEG). The fusion can be plated on a plate containing a medium that allows selective growth of the fused cells. Thereafter, the fused cells can be allowed to grow for about 8 days, and the supernatant from the resulting hybridoma is collected and added to a plate that has been first coated with goat anti-mouse Ig.¹²⁵I-Vpr (or¹²⁵A label such as (I-ANT) is added to each well, followed by incubation. Thereafter, positive wells can be detected by autoradiography. Positive clones can be grown in large scale cultures, after which the supernatant is purified on a protein A column (Pharmacia).
[0070]
The monoclonal antibodies of the present invention are described in Alting-Mees et al., "Monoclonal Antibody Expression Library: A Rapid Alternative to Hybridomas", Strategies In Molecular Biology, 3: 1-9 (1990), which is incorporated herein by reference. ) Can be manufactured using alternative techniques, such as the technique described in US Pat. Similarly, various binding partners can be constructed using recombinant DNA technology to incorporate the variable regions of the gene encoding the specific binding antibody. Such a technique is described in Larrick et al., Biotechnology, 7: 394 (1989).
[0071]
The monoclonal antibodies of the present invention include chimeric antibodies, for example, humanized versions of mouse monoclonal antibodies. Such humanized antibodies can be prepared by known techniques, and offer the advantage of reduced immunogenicity when the antibodies are administered to humans. In one embodiment, a humanized monoclonal antibody comprises the variable region of a murine antibody (or just its antigen binding site) and a constant region derived from a human antibody. Alternatively, a humanized antibody fragment can include the antigen binding site of a murine monoclonal antibody and a variable region fragment derived from a human antibody, which has no antigen binding site. Techniques for producing chimeric and further engineered monoclonal antibodies include Riechmann et al. (Nature, 332; 323, 1988), Liu et al. (PNAS, 84: 3439, 1987), Larrick et al. (Bio / Technology). 7: 934, 1989), and Winter and Harris (TIPS, 14: 139, May 1993). Techniques for making antibodies by gene transfer are described in UK Patent Nos. 2,272,440, 5,569,825 and 5,545,806, and related claims claiming priority therefrom. Patents, all of which are incorporated herein by reference, can be found.
[0072]
Once isolated and purified, antibodies to Vpr and ANT, as well as antibodies to other Vpr-binding proteins and ANT-binding proteins, are tested for the presence of Vpr and ANT in the sample using established assay protocols. Can be used to detect Further, the antibodies of the present invention can be used therapeutically to bind to Vpr or ANT and inhibit its activity in vivo.
[0073]
Antibodies to Vpr or ANT and other Vpr binding proteins or ANT binding proteins can be used to modulate the biological activity of Vpr and ANT. One class of these antibodies results in mitochondrial membrane permeabilization and apoptosis. In contrast, another class of these antibodies can inhibit mitochondrial membrane permeabilization and apoptosis.
[0074]
Such antibodies that can further block Vpr-ANT binding can be used to inhibit the biological activity resulting from such binding. Such blocking antibodies can be identified using any suitable assay technique, such as by testing the antibody for its ability to inhibit Vpr from binding to ANT. Alternatively, blocking antibodies can be identified in assays for the ability of Vpr to inhibit the biological effects that result from binding ANT in cells.
[0075]
Such antibodies can be used in in vitro methods, or can be administered in vivo to inhibit a biological activity mediated by the substance that produced the antibody. Various disorders are caused (directly or indirectly) or exacerbated (directly or indirectly) by the interaction of Vpr with ANT. Therapeutic methods include administering the blocking antibody to the mammal in vivo in an amount effective to inhibit ANT-mediated apoptosis. Monoclonal antibodies are generally preferred for use in such methods of treatment. In one embodiment, an antigen-binding antibody fragment is used.
[0076]
Antibodies can be screened for agonist (ie, mimic ligand) properties. Such antibodies, when bound to ANT, induce a biological effect similar to that induced when Vpr binds to ANT (eg, apoptosis). Agonist antibodies can be used to induce ANT-mediated apoptosis of cells.
[0077]
Provided herein are compositions comprising an antibody to Vpr or ANT and a physiologically acceptable diluent, excipient or carrier. Suitable components of such compositions are as described above for compositions containing Vpr or ANT.
[0078]
Also provided herein are conjugates comprising a detectable (eg, diagnostic) therapeutic agent conjugated to the antibody. Various examples of such agents are set forth above. Such conjugates are used in a variety of in vitro or in vivo approaches.
[0079]
Other molecules
The present invention also includes molecules that compete with Vpr binding to ANT or enhance Vpr binding to ANT. Such molecules can be identified by screening assays described herein, or by structure-based design using, for example, molecular modeling of Vpr-ANT binding.
[0080]
Pharmaceutical compositions and diagnostic compositions
The compositions of the present invention can contain peptide or non-peptidic molecules in any form, such as native proteins, variants, derivatives, oligomers and biologically active fragments. In specific embodiments, the composition comprises a soluble polypeptide or an oligomer comprising a soluble Vpr, Bcl-2 or ANT polypeptide or fragment.
[0081]
Provided herein are compositions comprising an effective amount of a molecule of the invention in combination with other ingredients such as a physiologically acceptable diluent, carrier or excipient. The molecules can be formulated according to known methods used to prepare pharmaceutically useful compositions. Molecules include pharmaceutically acceptable diluents (eg, saline, Tris-HCl, acetate and phosphate buffered solutions), preservatives (eg, thimerosal, benzyl alcohol, parabens), emulsifiers, It can be combined with the solubilizing agent, adjuvant and / or carrier, as the sole active substance, or in a mixture with other known active substances suitable for the given application. Suitable formulations for the pharmaceutical composition include those described in Remington's Pharmaceutical Sciences (16th edition, 1980, Mack Publishing Company, Easton, PA).
[0082]
Further, such compositions can be complexed with polyethylene glycol (PEG), metal ions, or formulated with polymeric compounds such as polyacetic acid, polyglycolic acid, hydrogels, dextrans, or liposomes. , Microemulsions, micelles, single-layer or multi-layer vesicles, erythrocyte ghosts or spheroblasts. Such compositions influence the physical state, solubility, stability, rate of in vivo release and rate of in vivo clearance, and are therefore chosen according to the intended application.
[0083]
The compositions of the present invention can be administered in any suitable manner, for example, topically, parenterally, or by inhalation. The term "parenteral" includes injection, for example, by subcutaneous or intravenous or intramuscular routes, and also includes local administration, for example, at the site of a disease or injury. Sustained release from the implant is also conceivable. One skilled in the art will recognize that suitable dosages will vary depending on factors such as the nature of the disorder being treated, the weight, age and general condition of the patient, and the route of administration. Preliminary doses can be determined according to animal studies, and dosage estimates for human administration are made according to art-recognized practices.
[0084]
Compositions comprising the nucleic acid in a physiologically acceptable formulation are also contemplated. DNA can be formulated, for example, for injection.
[0085]
Screening method for ANT changes
The invention also provides a method of screening for genetic or epigenetic changes in the expression or structure of three ANT isotypes in humans.
[0086]
The present invention provides for the diagnosis of diseases associated with abnormal ANT expression. For example, in certain cancers, certain changes in ANT may be associated with the cancer. Diagnosis of such cancers can be achieved, for example, using Vpr, as described herein, or using a fragment of Vpr that can retain binding to ANT in a binding assay. Thereby, the expression or structure of the three ANT isotypes in the patient can be revealed and a diagnosis can be achieved.
[0087]
Methods for specifically killing cells
Vpr or a biologically active fragment thereof (such as vpr52-96) can be used to induce apoptosis in cells. In one embodiment, the vpr52-96 polypeptide is fused to a molecule to target a particular cell type and induces apoptosis in such a cell type. In a further aspect, the conjugate of the Vpr targeting molecule specifically kills cancer cells. The methodology demonstrates that IL-2 receptor-bearing cells have been selectively killed in vitro using a recombinant chimeric protein containing an interleukin-2 (IL-2) protein fused to Bax. May be similar to R. Aquilan, S.M. Yarkoni and H.M. Lorberbum-Galski, FEBS Lett. 457: 271-6 (1999).
[0088]
In other embodiments, the conjugates of the biologically active Vpr targeting molecules can be used to specifically target other cell types involved in the disease and kill such cells. it can.
[0089]
Double exam
To study the role of ANT in apoptosis, and more particularly, the role of ANT in mitochondrial membrane permeabilization (Brenner et al., Oncogene, 2000; Costantini et al., Oncogene, 2000), we studied the anti-antagonism of ANT. A functional dual test has been developed that allows simultaneous measurement of port (life support) and pore (lethal) functions in artificial lipid bilayers or liposomes.
[0090]
The principle of this functional double test is the reconstitution of ANT in liposomes, the encapsulation of various substrates (fluorescent substrate and ATP) inside proteoliposomes, the addition of enzymes and ADP outside the liposomes, and It is based on the fluorescence measurement of salting out of the substrate through the pores formed by the ANT, and at the same time on the luminescence measurement of ATP transported in response to exogenous ADP. To demonstrate its effect on the two functions of ANT, any peptide or non-peptide compound can be reconstituted with ANT during liposome formation (eg, Bax, Bcl-2, Bcl-x (L )) Or can be encapsulated in liposomes or added to proteoliposomes in an exogenous manner (eg, addition of peptide molecules [eg, Vpr, Vpr52-96], Bld, etc.) or Not available (atractyloside, calcium, t-BHP, diamide, BA, cyclosporin A, verteporfin, etc.). Quantitative measurements can be made in a fixed point or kinetic manner. This test is performed in a 96-well microplate and can be adapted for HTS (High Throughput Screening).
[0091]
This functional duplicate test allows the screening of molecules that induce or inhibit apoptosis, the screening of ANT partner molecules that can convert or promote the conversion of ANT to the pore, or the specific functional form of ANT (life support function). And / or changes in lethal function, changes in the ratio of ANT isotypes). The antiport function test allows one to evaluate the toxicity of the molecule on the life support function of ANT.
[0092]
This specification is most thoroughly understood in light of the teachings of the references cited herein, which are incorporated herein by reference. The aspects and examples in this specification are illustrative of various aspects of the present invention, but should not be construed as limiting the scope of the invention. One skilled in the art will readily recognize that many other embodiments are encompassed by the present invention.
[0093]
Example 1
Physical and functional interaction between Vpr and ANT
Surface plasmon resonance measurement showed that the purified detergent-solubilized ANT protein was compatible with the C-terminal component biotin-Vpr52-96 of immobilized Vpr with an affinity in the nanomolar range (mutated biotin-Vpr52- 96 [R73, 80A] (FIG. 1A and B). This interaction is regulated by two ANT ligands that affect the conformation of ANT differently (M. Klingenberg, J. Membrane Biol. 56, 97-105 (1980)): binding of Vpr Atractyloside (Atr), a PTPC activator that is advantageous for, and boncrekinic acid (BA), a PTPC inhibitor that reduces Vpr binding (FIG. 1C). Vpr52-96 binding to the membrane is greatly enhanced in ANT reconstituted liposomes when compared to protein-free liposomes (FIG. 2A). This ANT action is inhibited by BA (FIG. 2B). Vpr52-96 (N-terminal component of Vpr [Vpr1-51] or variant Vpr52-96 (which is not arginine 77 replaced by alanine; Vpr52-96 [R77A])) are also ANT proteoliposomes. But has no effect on liposomes alone (FIG. 2C).
[0094]
A Bcl-2-like protein binds to the ANT motif (aa 105-155) (I. Marzo et al., Science, 281, 2027-2031 (1998)), and its involvement in regulating apoptosis is confirmed by ANT deletion mapping (MKA Bauer, A. Schubert, O. Rocks, S. Grimm, J. Cell Biol. 147, 1493-1501 (1999)). This motif partially overlaps the second ANT loop (aa 92-116), which is a regulatory domain exposed in the transmembrane space (G. Brandolin, A. Le-Saux, V. Trezeguet, G. J. Lauquinn, P. V. Vignais, J. Bioenerg. Biomembr. 25, 493-501 (1993); M. Klingenberg, J. Bioenerg. Biomembr. 25, 447-457 (1993)). The peptide corresponding to the overlap of this loop with the Bcl-2 binding motif (ANT104-116) is likely to interact with Vpr through a direct association with Vpr52-96 (inset in FIG. 1D). Was inhibited (FIG. 1C).
[0095]
Neither the topologically related peptide motif from the human phosphate carrier nor the mutant and scrambled forms of ANT104-116 (control peptide in FIG. 1C) had such an inhibitory effect. ANT104-116 (but not the control peptide) also prevented Vpr52-96 induced membrane permeation of ANT proteoliposomes (FIG. 2C). This indicates that in the context of a lipid bilayer, the action of Vpr was associated with a direct interaction with ANT. In planar lipid bilayers, low doses of Vpr52-96 (<1 nM) were unable to form channels unless ANT was present.
[0096]
ANT and Vpr52-96 together have different channels whose conductance (190 ± 2 pS) (FIG. 3) is much larger than the channel formed by the high dose (180 nM) of Vpr52-96 alone (55 ± 2 pS). (FIG. 3, and SC Piller, GD Ewart, A. Premkumar, GB Cox, PW Gauge, Proc. Natl. Acad. Sci. USA, 93, 111-115 (1996))^{2 +}Within the range of the channels formed by the treated ANT (N. Brustovesky, M. Klingenberg, Biochemistry, 35, 8483-8488 (1996); C. Brenner et al., Oncogene, 19, 329-336 (2000). ). These biophysical experiments demonstrate that ANT and Vpr interact directly within the membrane to form a functionally capable channel-forming hetero (poly) mer.
[0097]
Example 2
Oxidative properties of purified mitochondria exposed to Vpr
Purified mitochondria pre-incubated with Vpr52-96 (FIG. 4A, trace b) had significantly incomplete respiratory control (RC) when compared to untreated organelles (FIG. 4A, trace a). Vpr increases succinic acid oxidation preceding ADP addition and stimulates the inhibitory effects of oligomycin (a specific ATPase inhibitor) and uncoupling by the protonophorecarbamoyl cyanide m-chlorophenylhydrazone (CCCP) Both effects were disabled. Thus, Vpr52-96 (but not Vpr1-51) reduced RC (oxygen consumption ratio of oligomycin to CCCP) to a value of 1.1 compared to 5.3 in control mitochondria (FIG. 4B). The complete Vpr protein (Vpr1-96), and a short peptide (Vpr71-82) corresponding to the minimal "mitochondrial toxicity" motif of Vpr (LG Macleadie et al., Proc. Natl. Acad. Sci. USA, 92, 2770). IG Macleadie et al., FEBS Lett. 410, 145-149 (1997); E. Jacotot et al., J. Exp. Med. 191, 33-45 (2000)) also have RC values. (FIG. 4B). Notably, the loss of RC induced by Vpr was not associated with a significant decrease in the rate of oxidation (FIG. 4A). This suggests that a large loss of membrane bound cytochrome c did not occur when performing a short incubation with Vpr52-96. Therefore, addition of cytochrome c to Vpr52-96 treated mitochondria, which oxidizes succinic acid, did not stimulate the rate of oxygen uptake (FIG. 4A, trace b). The observation of Vpr-mediated uncoupling of the respiratory chain prompted us to test its ability to induce IM permeabilization. IM is essentially impermeable to NADH (P. Rustin et al., J. Biol. Chem. 271, 14785-14790 (1996)), so that when NADH is added to control mitochondria, Oxygen uptake could not be measured (FIG. 5A, trace a). However, the addition of Vpr52-96 promoted NADH-stimulated marked oxygen consumption (FIG. 5A, trace b). This indicates that Vpr increased the permeability of IM to both protons (which result in uncoupling; FIG. 4A, trace b) and NADH (FIG. 5A, trace b).
[0098]
The differential kinetics of the inner and outer MMPs for NADH and cytochrome c were each evaluated (FIG. 5B). NADH oxidation by mitochondria to which Vpr52-96 was added was found to be maximal after 10 minutes (Figure 5B). Under similar conditions, Vpr52-96 only induced less than adequate access of cytochrome c to cytochrome c oxidase (FIG. 5B). Furthermore, ΔΨ_mLoss occurred sufficiently before cytochrome c release could be detected by immunoblot (FIG. 5C). Thus, Vpr 52-96 produce a sufficiently inner MMP before the OM becomes permeable to exogenous cytochrome c. Thus, at the ultrastructural level (see below, FIG. 6C), mitochondria treated with Vpr 52-96 showed matrix swelling before rupture of the OM became apparent.
[0099]
Example 3
Bcl-2-mediated inhibition of Vpr action on mitochondria
Extracellular addition of Vpr to intact cells results in a rapid ΔΨ before nuclear condensation occurs._mWas induced. These effects were prevented by microinjecting recombinant Bcl-2 into the cytoplasm (FIG. 6A). Preincubation of purified mitochondria with recombinant Bcl-2 (or ANT ligand BA) also prevented Vpr-mediated inner MMPs to NADH (FIG. 6B). Concomitantly, Vpr-induced matrix swelling (FIG. 6C) and ΔΨ_mBoth loss (FIGS. 6D and E) were due to (Bcl-2Δα5 / 6 (deletion mutants lacking hypothetical pore-forming α5 and α6 helices, S. Schendel, M. Montal, J. C. 5, by Bcl-2 (as opposed to Cell Death Differ. 5, 372-380 (1998)) and by two pharmacological inhibitors of PTPC (BA and cyclosporin A; CsA) and specific VDAC inhibitors Koenigs polyamine (PA10; S. Stanley, JA Dias, DD 'Arcangelis, CA Mannella, J. Biol. Chem. 270, 16694-16700 (1995)). PA10 also produced Vpr52 against intact cells. Inhibits the action of 96. (FIG. 6A) Vpr52-96 binding to purified mitochondria was completely abolished by pre-incubation of organelles with PA10 and partially reduced by BA, but not by CsA. (FIG. 7B) Therefore, Vpr must approach mitochondria via VDAC.
[0100]
Bcl-2 prevents Vpr from crossing OM via VDAC (based on Bcl-2-mediated closure of VDAC) (S. Shimizu, M. Narita, Y. Tsujimoto, Nature, 399, 483). 487 (1999); S. Shimizu, A. Konishi, T. Kodama, Y. Tsujimoto, Proc. Natl. Acad. Sci. USA, 97, 3100-3105 (2001)), and / or (ANT) Inhibiting Vpr action on ANT (based on physical and functional interactions) (I. Marzo et al., Science, 281, 2027-2031 (1998); C. Brenner et al., Oncogene, 19, 329-336 ( 2000); ita et al, Proc.Natl.Acad.Sci.USA, 95,14681~14686 (1998)) can be expected. Recombinant Bcl-2 showed Vpr52-96 induced matrix swelling (FIG. 6C) and ΔΨ_mAlthough the loss (FIGS. 6D and E) was strongly reduced, the binding of Vpr52-96 to purified mitochondria could not be impaired (FIG. 7B). Differential inhibitory effects of PA10 and Bcl-2 on Vpr-ANT interaction were confirmed in different experimental systems. PA10 completely abolished affinity-mediated purification of ANT using biotinylated Vpr 52-96 (FIG. 7C). However, its effect was evaluated on intact mitochondria (in this case, Vpr 52-96 had to cross the OM to reach ANT). In contrast, PA10 did not affect Vpr52-96-mediated purification of ANT from Triton-solubilized mitochondria (where ANT could easily access Vpr52-96). Under the same conditions, despite its addition to intact or solubilized mitochondria, Bcl-2 reduced Vpr52-96 mediated recovery of ANT (FIG. 7C). Thus, Bcl-2 does not interfere with the VDAC-mediated groove (inhibited by PA10), which allows Vpr52-96 to cross the OM.
[0101]
Example 4
Bcl-2-mediated inhibition of Vpr-ANT interaction
In addition, a series of experiments showed that Bcl-2 regulates the physical and functional interaction of Vpr with ANT. Recombinant Bcl-2 (but not Bcl-2 Δα5 / 6) reduced Vpr52-96 binding to soluble ANT (FIG. 8A) or membrane-bound ANT (FIG. 8B). Since Bcl-2 did not bind to Vpr52-96, inhibition of Vpr / ANT binding appears to be due to a direct Bcl-2 / ANT interaction (I. Marzo et al., Science, 281, 2027). 2031 (1998); C. Brenner et al., Oncogene, 19, 329-336 (2000)). Thus, Bcl-2 abolished Vpr52-96-induced channel formation in the ANT-containing lipid bilayer. In contrast, under the same conditions, Bax deteriorated the conductance of the Vpr52-96-ANT channel to an average of 245 ± 2S (when compared to 190 ± 2 for Vpr52-96-ANT without any further addition). (FIG. 8C).
[0102]
Example 5
Double test protocol
1. Purification of ANT. ANT was purified from rat heart and reconstituted in liposomes according to the protocol described by Brenner et al., Oncogene, 2000.
[0103]
2. Reconstitution of ANT in liposomes. ANT (0.03 mg / ml) in the presence of 0.3% n-octyl-β-D-pyranoside in liposomes composed of phosphatidylcholine and cardiolipin (PC: CL; 45: 1; W; w; and lipids) (300 ng of ANT per mg) was incorporated for 2 minutes at room temperature. If necessary, other proteins or compounds were added at this stage during uptake (eg, Bcl-2, Bax). The detergent was then removed by dialyzing the liposomes overnight at 4 ° C. against a buffer of HEPES @ 10 mM, sucrose 125 mM, pH 7 (eg, about 1 liter buffer per ml liposome).
[0104]
3. Pore function test. Liposomes were loaded with 4-UMPＵ1 mM (4-methylumbelliferyl phosphate) in KCl 10 mM by sonication (50 W, 22 seconds). Thereafter, the proteoliposomes were separated on a Sephadex @ G25 column to remove unencapsulated compounds. Elution was performed using a buffer of HEPES @ 10 mM, sucrose 125 mM, pH 7, at room temperature. In a 96-well microplate, 25 μl of liposomes were placed in each well.
[0105]
25 μl of the product to be tested was added and incubated for 30 minutes to 60 minutes with various compounds at room temperature (eg, 30 minutes for Vpr 52-96; 60 minutes for non-peptide compounds). Thereafter, alkaline phosphatase (5 U / ml) and 150 μl of HEPES @ 10 mM, sucrose 125 mM, MgCl 2 @ 0.5 mM, pH 7.4 reaction buffer were added. Plates were incubated for 15 minutes with shaking at 37 ° C. to allow enzymatic conversion of 4-UMP to 4-umbelliferone (4-UM). The reaction was stopped by the addition of 150 μl stop buffer (10 mM @ HEPES-NaOH, 200 mM @ EDTA, pH 10). The measurement was performed by 4-UM fluorescence measurement (excitation: 365 nm; emission: 450 ± 5 nm). In each experiment, an untreated sample of liposomes, an encapsulated sample of 4-UMP, and a maximum salting out sample of 4-UMP were made, and the results are expressed as a percentage of salted out 4-UMP. be able to.
[0106]
4) Antiport function test. The liposomes were loaded with ATP @ 1 mM (4-methylumbelliferyl phosphate) in KCl @ 10 mM by sonication (50 W, 22 seconds). Thereafter, the proteoliposomes were separated on a Sephadex @ G25 column to remove unencapsulated compounds. Elution was performed at room temperature using a small amount of 10 mM HEPES buffer solution, 125 mM sucrose, pH7. In a 96-well microplate, 25 μl of liposomes were placed in each well.
[0107]
25 μl of the product to be tested was added and incubated at room temperature for 30-60 minutes (eg, 30 minutes for Vpr 52-96; 60 minutes for non-peptide compounds). Then, 25 μl of luciferase (HS II Boerchinger kit) was added, and the emitted light was measured immediately. The results are expressed as a percentage by comparison to the maximum ATP exposed in response to the addition of 400 μl to the outside of the liposome.
[0108]
5. Remarks. Proteoliposomes loaded with 4-UMP and KCl for the purpose of measuring pore function are frozen at −20 ° C., whereas proteoliposomes loaded with ATP and KCl are not frozen for measuring transporter function .
[0109]
[Table 1]

[Brief description of the drawings]
FIG. 1A
FIG. 1 illustrates the physical and functional interaction between Vpr and ANT.
A. Plasma surface resonance sensorgrams of ANT interaction with Vpr52-96, Vpr52-96 [R73A, 80A] or an unrelated control (Co). Only sensorgrams of interaction with Vpr52-96 show increased binding as a function of time and a positive signal at the onset of the dissociation phase (off). Calculated K of interaction_D(K_D= K_on/ K_off) Is 9.7 ± 6.4 nM (X ± SD, n = 5).
FIG. 1B
FIG. 1 illustrates the physical and functional interaction between Vpr and ANT.
B. Langmuir isotherms measured at different concentrations of ANT in sensorgrams corrected by blank subtraction (sensorgrams were obtained with Vpr 52-96 [R73A, 80A]).
FIG. 1C
FIG. 1 illustrates the physical and functional interaction between Vpr and ANT.
C. Modulation of Vpr52-96-ANT interaction by ANT ligand and ANT-derived peptides. Measurements are made as in A in the absence (φ) or in the presence of boncrequinic acid (BA, 250 μM), atractyloside (Atr, 50 μM), ANT104-116 peptide or three control peptides (all 5 μM) Was. ANT-2-derived peptide ANT104-116 [DKRTQFWRYFAGN] and control peptide (Co.I: scrambled ANT104-116 [FQNYWGHKRFRDA]; Co.II: mutant ANT104-116 [DGHKKFWWGYFAGN]; Co.III: ANT-related human phosphorus Topologically equivalent peptides (aa 149-161) derived from the acid transporter protein [SNMLGEENTYLWR]._0a/ K₀) × 100 (where k is_0aAnd k₀Is the initial velocity in the presence or absence of the drug, respectively).
FIG. 1D
FIG. 1 illustrates the physical and functional interaction between Vpr and ANT.
D. Langmuir isotherm for binding of ANT104-116 to biotinylated Vpr52-96 (as measured in A). Calculated K of interaction_DIs 35 μM.
FIG. 1E
FIG. 1 illustrates the physical and functional interaction between Vpr and ANT.
E. FIG. It is the schematic which shows the topology of ANT and the peptide ANT104-116 derived from ANT-2.
FIG. 2A
FIG. 2 shows physical interaction (A, B) and functional interaction (C) between Vpr and ANT-containing liposomes.
A. Dose response curves of FITC-labeled Vpr52-96 binding to ANT-liposomes and liposomes alone.
FIG. 2B
FIG. 2 shows physical interaction (A, B) and functional interaction (C) between Vpr and ANT-containing liposomes.
B. Binding of FITC-Vpr52-96 (2 μM) to liposomes alone, ANT-proteoliposomes in the presence or absence of BA (50 μM).
FIG. 2C
FIG. 2 shows physical interaction (A, B) and functional interaction (C) between Vpr and ANT-containing liposomes.
C. Permeabilization of ANT proteoliposomes by Vpr (X ± SD, n = 3). The liposomes are loaded with 4-methylumbelliferyl phosphate (4-MUP), and BA (50 μM), ADP (800 μM) and / or the indicated peptide (0.5 μM, Vpr52- (Preincubated with 95 for 5 minutes) in the presence or absence of Atr (200 μM) or the indicated Vpr-derived peptide (1 μM) for 80 minutes. Thereafter, alkaline phosphatase was added to convert the 4-MUP released from the liposome into the fluorescent dye 4-methylumbelliferone (4-MU), and the rate of 4-MUP release induced by the Vpr-derived peptide was determined as " Calculated as described in Materials and Methods.
FIG. 3A
FIG. 3 depicts the electrophysiological properties of Vpr52-96 and ANT in planar lipid bilayers. Current histograms of Vpr52-96 (80 nM, +150 mV), Vpr52-96 (0.4 nM, +100 mV), ANT (1 nM, +110 mV) and Vpr52-96 + ANT (0.4: 1 nM, +115 mV), and associated histograms of conductance levels (Right) is shown.
A. Synergy of ANT and Vpr 52-96 at single channel level. Vpr52-96 (80 nM, +150 mV), Vpr52-96 (0.4 nM, +100 mV), ANT (1 nM, +110 mV) and Vpr52-96 + ANT (0.4: 1 nM, +115 mV) after being incorporated into the synthetic membrane. Current fluctuation. Single channel recordings were made using "Tip-Dip" technology. The records shown are representative of at least three independent measurements.
FIG. 3B
FIG. 3 depicts the electrophysiological properties of Vpr52-96 and ANT in planar lipid bilayers. Current histograms of Vpr52-96 (80 nM, +150 mV), Vpr52-96 (0.4 nM, +100 mV), ANT (1 nM, +110 mV) and Vpr52-96 + ANT (0.4: 1 nM, +115 mV), and associated histograms of conductance levels (Right) is shown.
B. Statistical analysis of conductance obtained in A. The results were expressed as current variations at various voltages. Conductance (γ; in pico-Siemens (pS)) is calculated by dividing current by voltage.
FIG. 4A
FIG. 4 shows the oxidative properties of purified mitochondria exposed to Vpr.
A. Oxygen consumption curves after addition of the indicated drugs. Trace a: control mitochondria (no pretreatment). Trace b: mitochondria pretreated with 1 μM Vpr 52-96 for 10 minutes. The numbers along the trajectory indicate the O consumed for 1 mg protein per minute.₂Nmol.
FIG. 4B
FIG. 4 shows the oxidative properties of purified mitochondria exposed to Vpr.
B. Respiratory control (RC) calculated by dividing the oxygen consumption in the presence of CCCP 10 minutes after the addition of 1 μM of the Vpr-derived peptide by the oxygen consumption measured with oligomycin (measured as A). ) Value (average of three measurements).
FIG. 5A
FIG. 5 shows inner versus outer mitochondrial membrane permeabilization.
A. Respiratory measurements made after addition of NADH and Vpr 52-96 (1 μM). The numbers along the trajectory indicate the O consumed for 1 mg protein per minute.₂Nmol. NApr-independent O stimulated by Vpr₂Note that the consumption was completely sensitive to rotenone.
FIG. 5B
FIG. 5 shows inner versus outer mitochondrial membrane permeabilization.
B. Kinetics of Vpr52-96 induced inner membrane permeabilization for NADH and reduced outer membrane permeation kinetics for cytochrome c. Oxygen consumption was measured as in case A (traces CD) in the presence of 2 mM NADH (closed squares) and oxidation of cytochrome c (15 μM) (open circles) was described (Rustin et al., 1994). Was measured by spectrofluorimetry as follows. The 100% value of cytochrome c oxidation was determined by the addition of 2.5 mM lauryl maltoside.
FIG. 5C
FIG. 5 shows inner versus outer mitochondrial membrane permeabilization.
C. ΔΨ induced by Vpr 52-96_mKinetics of loss and cytochrome c release. The purified mitochondria were treated with 1 μM Vpr52-96, and ΔΨ_mUsing the sensitive fluorescent dye JC-1, low ΔΨ_mThe ratio of mitochondria having the following was used for measurement by cytofluorimetry. At the same time, cytochrome c was immunodetected in the mitochondrial supernatant.
FIG. 6A
FIG. 6 shows Bcl-2-mediated inhibition of Vpr action on mitochondria.
A. ΔΨ of Vpr52-96 induction induced in intact cells_mDisappearance. COS cells were microinjected only with recombinant human Bcl-2 (10 μM), Konigs polyanion (PA, 2 μM) or PBS, and then incubated for 3 hours in the absence (Co.) or presence of 1 μM Vpr52-96. And ΔΨ_mSensitive dye JC-1 (2 μM; ΔΨ_mMitochondrial red fluorescence, ΔΨ_mBut with low mitochondrial green fluorescence).
FIG. 6B
FIG. 6 shows Bcl-2-mediated inhibition of Vpr action on mitochondria.
B. Effect of Bcl-2 on Vpr-induced inner MMPs on NADH. Mitochondria were left untreated (Co.) or pretreated (10 min) with Bcl-2 (0.8 μM) or BA (10 μM). Oxygen consumption of purified mitochondria was measured as in FIG. 5 after addition of succinate + CCCP or NADH as indicated.
FIG. 6C
FIG. 6 shows Bcl-2-mediated inhibition of Vpr action on mitochondria.
C. Ultrastructural effects of Vpr on isolated mitochondria. Electron micrographs were obtained after pre-incubation (5 min) with 0.8 μM Bcl-2 or 2 μM PA10, followed by incubation of mitochondria with 3 μM Vpr 52-96 for 5 or 15 min.
FIG. 6D
FIG. 6 shows Bcl-2-mediated inhibition of Vpr action on mitochondria.
D. ΔΨ by Vpr52-96 induction in purified mitochondria_mEffect of Bcl-2 and PA-10 on elimination. Preincubating the isolated mitochondria (200 μg protein / ml) with the indicated inhibitors (5 μM CsA, 50 μM BA, 0.8 μM Bcl-2, 2 μM PA10; 5 min to 10 min) After washing (10 minutes, 6800 g, 20 ° C.), ΔΨ_mIncubation with the sensitive dye JC-1 (200 nM, 10 min) and exposure to Vpr 52-98 (3 μM, 5 min) were subjected to fluorescence (570-595 nm) and particle size (FSC) flow cytometry measurements. The number is about 10⁴The percentage of JC-1 high mitochondria and JC-1 low mitochondria among events is shown.
FIG. 6E
FIG. 6 shows Bcl-2-mediated inhibition of Vpr action on mitochondria.
E. FIG. Quantification of the frequency of JC-1 low mitochondria after incubation with various Vpr-derived peptides (X ± SD, n = 5). Purified mitochondria were purified with Bcl-2 (0.8 μM), Bcl-2Δα in PT buffer._{5 / 6}(0.8 μM) or BA (10 μM) in the presence or absence for 10 minutes, and ΔΨ_mIncubate with the sensitive dye JC-1 (200 nM, 10 min) followed by 3 μM Vpr52-96 (wt, biotinylated or modified as shown) for 5 min, or 5 μM-10 μM Vpr1-96, Vpr1 -51, Vpr71-96, Vpr71-82 (wt or modified as indicated) for 10 minutes and finally subjected to flow cytometry analysis as in D.
FIG. 7A
FIG. 7 depicts the differential effects of Bcl-2 and PA-10 on Vpr52-96 binding to mitochondria.
A. Vpr 52 to 96 are ΔΨ_mBinds to mitochondria before inducing loss. Mitochondria were either left unstained (inset at Co.) or ΔΨ._mThe insensitive mitochondrial dye MitoTracker® Green (75 mM), alone (MTG) or with 0.5 μM FITC-Vpr 52-95 (green fluorescence)_mExposure was performed in combination with the sensitive mitochondrial dye MitoTracker® Red (CMXRos; red fluorescence), followed by two-color analysis by flow cytometry. The numbers indicate the ratio of mitochondria in each quadruplicate.
FIG. 7B
FIG. 7 depicts the differential effects of Bcl-2 and PA-10 on Vpr52-96 binding to mitochondria.
B. PA-10, but not Bcl-2, inhibits binding of Vpr52-96 to mitochondria. Mitochondria are pre-incubated with the indicated inhibitors for 10 minutes, and the percentage of mitochondria labeled with FITC-Vpr 52-96 is measured as in A. C. Inhibitory effect of Bcl-2 on affinity purification of ANT by biotinylated Vpr52-96. Mitochondria were incubated with the indicated inhibitors and then exposed for 30 minutes at RT with 5 μM biotinylated Vpr 52-96. Mitochondria were dissolved after incubation (top) or before incubation (bottom) with biotinylated Vpr52-96 with Tris / HCl as described in Materials and Methods. Biotinylated Vpr52-96 complexed with the mitochondrial ligand was retained on avidin-agarose and subjected to ANT immunoblot detection.
FIG. 7C
FIG. 7 depicts the differential effects of Bcl-2 and PA-10 on Vpr52-96 binding to mitochondria.
FIG. 8A
FIG. 8 depicts Bcl-2-mediated inhibition of Vpr-ANT interaction.
A. Plasmon surface resonance measurements of Bcl-2-mediated inhibition of the interaction of Vpr52-96 with purified native ANT. Interactions were indicated at the indicated concentrations of recombinant Bcl-2, Bcl-2Δα._{5 / 6}Alternatively, it was measured after adding the recombinant Bid. Data (X ± SD, n = 3) was calculated as in FIG.
FIG. 8B
FIG. 8 depicts Bcl-2-mediated inhibition of Vpr-ANT interaction.
B. Effect of Bcl-2 on Vpr binding to ANT proteoliposomes. Retention (X ± SD, n = 3) of FITC-labeled Vpr52-96 in ANT proteoliposomes preincubated with 800 nM Bcl-2 or 2 μM PA10 was evaluated as in FIG. 2A.
FIG. 8C
FIG. 8 depicts Bcl-2-mediated inhibition of Vpr-ANT interaction.
C. Effect of Bcl-2 on Vpr-ANT channel formation in planar lipid bilayers. Single channel recordings (+75 mV) and corresponding amplitude histograms of Vpr52-96 + ANT + Bax (0.4: 1: 0.3 nM) and Vpr52-96 + ANT + Bcl-2 (0.4: 1: 1 nM) are shown. Control values for Vpr52-96 + ANT alone are as in FIG. 1e (not shown) (c, closed, o, open).
FIG. 9
FIG. 9 depicts a model of the Vpr / PTPC interaction. Vpr crosses the outer membrane through VDAC, which is inhibited by Koenigs polyanine. Vpr then interacts with ANT. Bcl-2 and the ANT ligand (boncrekinate) inhibit Vpr from binding to ANT, whereas CsA inhibits ANT pore formation via its action on cyclophilin D (Cyp-D). Indirectly affects function.

Claims (23)

A method for preventing the interaction between Vpr and ANT,
(A) providing a molecule capable of preventing full-length Vpr from binding to ANT; and (b) contacting the molecule with an ANT fragment, wherein the molecule comprises an ANT fragment and Vpr. A way to prevent interaction. 2. The method of claim 1, wherein the method prevents channel formation in the mitochondrial membrane. 2. The method of claim 1, wherein the method prevents permeabilization of the mitochondrial membrane. 2. The method of claim 1, wherein the method prevents cell death. 5. The method of claim 4, wherein the method prevents cell death by apoptosis. 2. The method of claim 1, wherein said molecule is Bcl-2 or a fragment thereof. A method for screening for a molecule that competes with the binding of the C-terminal portion of Vpr to ANT,
(A) providing a Vpr fragment capable of binding to ANT;
(B) contacting the Vpr fragment with an ANT fragment capable of binding Vpr in the presence and absence of a test molecule; and (c) contacting the ANT fragment with the ANT fragment in the presence and absence of the test molecule. A method comprising detecting binding of a Vpr fragment. 8. The method of claim 7, wherein said fragment comprises full length Vpr. 8. The method of claim 7, wherein said fragment comprises amino acids 52-96 of HIV-1 Vpr. A method of screening for a molecule that mimics Vpr or a Vpr fragment in its ability to physically interact with ANT,
(A) providing Vpr or a Vpr fragment capable of interacting with ANT;
(B) contacting the Vpr or Vpr fragment with a Vpr or an ANT fragment capable of interacting with the Vpr fragment in the presence or absence of the test molecule; and (c) in the presence or absence of the test molecule. Detecting the binding of said Vpr or Vpr fragment to said ANT fragment in. Peptidic or non-peptidic molecules that prevent permeabilization of the mitochondrial membrane and prevent Vpr from binding to ANT. Peptidic or non-peptidic molecules that cause permeabilization of the mitochondrial membrane and enhance Vpr binding to ANT. A pharmaceutical or diagnostic composition comprising a molecule according to claim 11. 14. A method for causing or preventing permeation of a mitochondrial membrane, comprising administering to a patient a composition according to claim 13. A method of screening for a genetic or epigenetic change in the expression or structure of three ANT isoforms in a human, comprising:
(A) providing a fragment of Vpr capable of binding to ANT together with a sample containing human ANT;
(B) mixing the fragment with a biological sample containing human ANT;
(C) mixing the fragment with a control sample containing human ANT;
(D) detecting the binding of Vpr to ANT in the biological sample and the control sample;
(E) correlating differences in binding with genetic or epigenetic changes in the ANT, and (f) optionally detecting differences in the ability of the ANT to form channels in liposomes or planar lipid bilayers. Method. A method for quantifying the level of three human ANT isoforms in a cell, comprising:
A method comprising: (a) mixing Vpr with a biological sample containing ANT in an amount effective to bind ANT; and (b) quantifying the level of Vpr binding to ANT. CLAIMS 1. A method for screening active molecules aimed at inducing or preventing the formation of lethal pores by ANT,
(A) providing purified ANT in an artificial lipid bilayer or liposome;
A method comprising: (b) contacting the molecule of interest to be screened with the ANT; and (c) detecting the formation of a lethal pore by measuring the release of a labeled substrate. A method for screening an active molecule for the purpose of inhibiting the formation of a lethal pore without hindering an antiport function,
(A) providing a composition comprising purified ANT in an artificial lipid bilayer or liposome with molecules that induce the formation of lethal pores;
(B) contacting the composition in the presence or absence of a test molecule;
A method comprising (c) detecting the presence of an antiport function by fluorescence, and (d) detecting, by another fluorescence, a test molecule that inhibits the formation of a lethal pore. 19. The method for screening an active molecule of interest according to claim 18, wherein in step a), the active molecule that induces formation of a lethal pore is Vpr, a Vpr fragment or a Vpr mutant. 19. The method for screening for an active molecule of interest according to claim 18, wherein in step a), the active molecule that induces the formation of a lethal pore is selected from the group comprising atractyloside, mastoparan, terbutyl or diamide. . 19. The method for screening for an active molecule of interest according to claim 18, wherein in step a), the active molecule that induces formation of a lethal pore is selected from the group of pro-apoptotic molecules of the Bcl-2 family. 19. The active molecule of interest according to claim 18, wherein in step a) the active molecule that induces the formation of a lethal pore is a BAX molecule selected from the group of pro-apoptotic molecules of the Bcl-2 family. Screening method. An isolated or purified peptide having the sequence DRHKQFWRYFAGN.

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